Methods of use of cd24 for the prevention and treatment of graft versus host disease and mucositis

ABSTRACT

The present invention relates to the use of a CD24 protein for preventing or treating Graft versus Host Disease and mucositis.

FIELD OF THE INVENTION

The present invention relates to compositions and methods for preventingand treating Graft versus Host Disease and mucositis.

BACKGROUND OF THE INVENTION

Allogeneic hematopoietic stem cell transplantation (HCT) is the onlyestablished curative therapy for a broad spectrum of high risk leukemiaand myelodysplasia in adults. An important function of allogeneictransplantation is to use donor T cells to eliminate allogeneic leukemiacells, this effect is called GVL. However, Graft vs host disease (GVHD)is a life threatening complication that occurs when the immune competentcells from the donor stem cell graft mount an immune attack against thehost. Activated donor T cells damage host epithelial cells following aninflammatory cascade that begins with the preparative regimen. The exactrisk is dependent on the stem cell source, age of the patient,conditioning, and GVHD prophylaxis used. The incidence is directlyrelated to the degree of human leukocyte antigens (HLA) disparity. Themedian onset of acute GVHD is typically 21 to 25 days aftertransplantation. The incidence ranges from 30-65% in recipients of fullyhistocompatible related donor transplants to 60% to 80% in recipients ofmismatched hematopoietic cells or hematopoietic cells from an unrelateddonor. Umbilical cord-blood transplantation has been associated withslower neutrophil recovery with lower incidence and later onset of acuteGVHD. Factors that increase the incidence include use of peripheralblood rather than bone marrow as the source of hematopoietic cells andolder recipient age. The median time of diagnosis of chronic GVHD is 4.5months after HLA-identical sibling transplantation and 4 months afterunrelated donor transplantation. De novo chronic GVHD almost neveroccurs after 2 years following allogeneic HCT.

For over 20 years, the combination of a calcineurin inhibitor (e.g.cyclosporine and tacrolimus) with methotrexate has remained the standardof care for the prevention of GVHD. Despite routine administration ofimmune prophylaxis, clinically significant GVHD (Grade II-IV) occurs inapproximately 30 to 65% of patients undergoing HLA matched related HCTand 60 to 80% of patients receiving unrelated donor HCT. Acute GVHD isan early event after HCT, with a median time to onset of approximately25 to 30 days. In patients with very severe GVHD, mortality rates exceed90%. One explanation for this is that, once established, ineffectiveresponses occur to front-line therapy with high dose corticosteroids ingreater than 50% of patients. Survival is significantly diminished forpatients who demonstrate steroid refractoriness or who require prolongedtreatment. Even when successful, high doses of corticosteroids are amajor source of morbidity due to increased infections and deconditioningthat places patients at significant risk for TRM.

Host tissue injuries caused by the HCT conditioning regimens, includinghigh-dose chemotherapy and/or total body irradiation (TBI), areconsidered to be the first step in the development of acute GVHD. Hosttissue injuries caused by the conditioning regimen lead to the releaseof proinflammatory cytokines (such as TNF-α, IL-1β and IL-6), and alsothe release of damage-associated molecular patterns (DAMPs) andpathogen-associated molecular patterns (PAMPs). Both DAMPs and PAMPs canactivate antigen-presenting cells (APCs), such as dendritic cells (DCs),by binding to pattern recognition receptors (PRRs). The host APCssubsequently activate donor T cells and an immunologic cascade thatresults in the release of pro-inflammatory cytokines and expansion ofthe antigen specific allo-reactive T cells that target host tissues,resulting in GVHD. It is therefore of great interest to explore whetherGVHD can be attenuated by targeting host response to tissue injuries andpreventing activation of APCs, the key processes in the initiation ofGVHD.

To date, treatment and prevention of GVHD has predominantly focused oneither pharmacologic inhibition or depletion of T cells through in vivoor ex vivo approaches to limit expansion of alloreactive T cells thatmediate tissue injury. While non-selective T-cell depleting strategies(e.g. antithymocyte globulin) are efficacious in preventing GVHD, theydo not improve survival due to offsetting risks for relapse, infectionand graft rejection. Conversely, more selective inhibition by targetingsingle pro-inflammatory cytokines has not demonstrated clinical benefitin treating GVHD. As a result, apart from antibodies that deplete Tcells, no biologics have been approved for GVHD and the combination oftacrolimus with methotrexate has remained the standard of care for theprevention of GVHD. The significant unmet medical needs call for moreselective biological products for both prophylaxis and treatment ofGVHD.

Mucositis is a common and painful side effect of chemotherapy andradiotherapy treatment for cancer that results in inflammation andulceration of the mucous membranes lining the digestive tract. It is aresult of tissue injury caused by the radiation/radiotherapy (RT) orchemotherapy. Mucositis can occur anywhere along the gastrointestinal(GI) tract, but oral mucositis refers to the particular inflammation andulceration that occurs in the mouth. Oral and gastrointestinal (GI)mucositis affects almost all patients undergoing high-dose chemotherapywith cytarabine and high-dose 5-fluorouracil, alkylating agents andplatinum based compounds and the majority of patients with malignanciesof the head and neck receiving radiotherapy. Radiation induced oralmucositis (RIOM) occurs in 100% of altered fractionation radiotherapyhead and neck cancer patients. Patients suffering from oral mucositisexperience severe pain, inflammation, ulceration, and bleeding that cansignificantly impede the patient's ability to swallow, which can alsolimit tumor control due to interruption of cancer treatment.Accordingly, oral mucositis is an important adverse effect seen incancer patients on chemotherapy and/or radiation therapy for the headand neck. Alimentary tract mucositis increases mortality and morbidityand contributes to rising health care costs. In the United Sates, theeconomic cost of RIOM was estimated to reach 17,000.00 USD per patientwith head and neck cancers.

The most profound mucositis burden is experienced as a result of theconditioning regimens used for hematopoietic stem cell transplant (HCT).The treatment regimen for HCT includes a pre-conditioning regimen,including highly mucotoxic chemotherapy with or without total bodyirradiation (TBI), which is required to kill off the recipient'scancerous hematopoietic cells prior to transplant. This preconditioningtreatment leads to serious damage throughout the alimentary tract. Lowgrade mucositis includes erythema of the mucosa and patchy ulcerationsor pseudomembranes. Severe mucositis (grade≥3) is associated withconfluent ulcerations or pseudomembranes and bleeding with minor trauma,which can progress to tissue necrosis, significant spontaneous bleedingand life-threatening consequences. Another indication related tomucositis in patients receiving HCT is oral graft-versus-host disease(GVHD), which is a form of chronic GVHD. As with chemotherapy andradiation induced mucositis, oral GVHD includes mucosal erythema,ulcerations, and painful desquamative oral lesions. However, a trueclinical case definition of oral acute GVHD is lacking, as severalfactors, particularly the conditioning chemotherapy with or withoutconcurrent radiation, contribute to oral lesion development during thefirst 28 days following HCT.

As indicated, GVHD, mucositis and related indications all involve anelement of tissue damage. To combat these challenges, there is a need inthe art for methods of effectively treating and preventing GVHD andmucositis.

SUMMARY OF THE INVENTION

Provided herein is a method of preventing or treating Graft versus HostDisease (GvHD) or mucositis in a subject in need thereof, which maycomprise administering a CD24 protein to the subject. Also providedherein is use of a CD24 protein in the manufacture of a medicament forpreventing or treating GvHD or mucositis in a subject. The method or usemay reduce the subject's risk of Grade III-IV acute GvHD. The subjectmay be human. The subject may undergo or may have undergone ahematopoietic stem cell transplantation (HCT). The subject may havecancer, which may be Acute Myeloid Leukemia (AML), Acute LymphoblasticLeukemia (ALL), Chronic Myelogenous Leukemia (CML), Myelodysplasticsyndrome (MDS), or Chronic Myelomonocytic Leukemia (CMML).

The CD24 protein may be administered at a dose of 240 mg or 480 mg. TheCD24 protein may be administered before or after the HCT, and may beadministered one day before the HCT. The CD24 protein may beadministered more than once, and may be administered in biweekly doses.The doses may comprise a dose on the day before the HCT, a dose on day14 after the HTC, and a dose on day 28 after the HCT, and the doses maybe, respectively, 480 mg, 240 mg, and 240 mg.

The CD24 protein may comprise a mature human CD24 polypeptide fused atits N-terminus or C-terminus to a Fc region of a mammalianimmunoglobulin (Ig) protein. The mature human CD24 polypeptide maycomprise the sequence set forth in SEQ ID NO: 1 or 2. The Ig protein maybe human. The Fc region may comprise a hinge region and CH2 and CH3domains of IgG1, IgG2, IgG3, IgG4, or IgA. The Fc region may comprise ahinge region and CH2, CH3 and CH4 domains of IgM. The CD24 protein maycomprise the sequence set forth in SEQ ID NO: 6, 11, or 12. The aminoacid sequence of the CD24 protein may consist of the sequence set forthin SEQ ID NO: 6, 11, or 12. The CD24 protein may be soluble, and may beglycosylated. The CD24 protein may be prepared using a eukaryoticexpression system, which may comprise expression from a vector inmammalian cells. The cells may be Chinese Hamster Ovary cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the amino acid composition of the full length CD24 fusionprotein, CD24Fc (also referred to herein as CD24Ig) (SEQ ID NO: 5). Theunderlined 26 amino acids are the signal peptide of CD24 (SEQ ID NO: 4),which are cleaved off during secretion from a cell expressing theprotein and thus missing from the processed version of the protein (SEQID NO: 6). The bold portion of the sequence is the extracellular domainof the mature CD24 protein used in the fusion protein (SEQ ID NO: 2).The last amino acid (A or V) that is ordinarily present in the matureCD24 protein has been deleted from the construct to avoidimmunogenicity. The non-underlined, non-bold letters are the sequence ofIgG1 Fc, including the hinge region and CH1 and CH2 domains (SEQ ID NO:7). FIG. 1B shows the sequence of CD24^(v)Fc (SEQ ID NO: 8), in whichthe mature human CD24 protein (bold) is the valine polymorphic variantof SEQ ID NO: 1. FIG. 1C shows the sequence of CD24^(A)Fc (SEQ ID NO:9), in which the mature human CD24 protein (bold) is the alaninepolymorphic variant of SEQ ID NO: 1. The various parts of the fusionprotein in FIGS. 1B and 1C are marked as in FIG. 1A and the variantvaline/alanine amino acid is double underlined.

FIG. 2 shows amino acid sequence variations between mature CD24 proteinsfrom mouse (SEQ ID NO: 3) and human (SEQ ID NO: 2). The potentialO-glycosylation sites are bolded, and the N-glycosylation sites areunderlined.

FIGS. 3A-C. WinNonlin compartmental modeling analysis ofpharmacokenitics of CD24IgG1 (CD24Fc). The opened circles represent theaverage of 3 mice, and the line is the predicted pharmacokinetic curve.FIG. 3A. i.v. injection of 1 mg CD24IgG1. FIG. 3B. s.c. injection of 1mg CD24IgG1 (CD24Fc). FIG. 3C. Comparison of the total amounts ofantibody in the blood as measured by areas under curve (AUC), half-lifeand maximal blood concentration. Note that overall, the AUC and Cmax ofthe s.c. injection is about 80% of i.v. injection, although thedifference is not statistically significant.

FIGS. 4A-B. CD24-Siglec G (10) interaction discriminates between PAMPand DAMP. FIG. 4A. Host response to PAMP was unaffected by CD24-Siglec G(10) interaction. FIG. 4B. CD24-Siglec G (10) interaction represses hostresponse to DAMP, possibly through the Siglec G/10-associated SHP-1.

FIGS. 5A-C. CD24 Fc binds to Siglec 10 and HMGB1 and activates Siglec G,the mouse homologue of human Siglec 10. FIG. 5A. Affinity measurement ofthe CD24Fc-Siglec 10 interaction. FIG. 5B. CD24Fc specifically interactswith HMGB-1 in a cation-dependent manner CD24Fc was incubated with HMGB1in 0.1 mM of CaCl₂ and MgCl₂, in the presence or absence of the cationchelator EDTA. CD24Fc is pulled down with protein G-beads, and theamounts of HMGB1, CD24Fc or control Fc is determined by Western blot.FIG. 5C. CD24Fc activates mouse Siglec G by inducing Tyrosinephosphorylation (middle panel) and association with SHP-1 (upper panel).The amounts of Siglec G are shown in the lower panel. CD24^(-/-) spleencells were stimulated with 1 μg/ml of CD24Fc, control Fc or vehicle(PBS) control for 30 minutes. Siglec G was then immunoprecipitated andprobed with anti-phospho-tyrosine or anti-SHP-1.

FIGS. 6A-B. CD24Fc inhibits production of TNF-α and IFN-γ by anti-CD3activated human T cells. The human PBML were stimulated with anti-CD3for 4 days in the presence or absence of CD24Fc and the amounts of IFN-γand TNF-α released in the supernatant of cell culture were measured byELISA. Data shown are means of triplicate. Error bar, SEM.

FIGS. 7A-B. CD24 inhibits inflammatory cytokine production by humanmacrophages. FIG. 7A. ShRNA silencing of CD24 leads to spontaneousproduction of TNF-α, IL-1β, and IL-6. THP1 cells were transduced withlentiviral vectors encoding either scrambled or two independent CD24shRNA molecules. The transduced cells were differentiated intomacrophages by culturing for 4 days with PMA (15 ng/ml). After washingaway PMA and non-adherent cells, the cells were cultured for another 24hours for measurement of inflammatory cytokines, by cytokine beadsarray. FIG. 7B. As in FIG. 7A, except that the given concentration ofCD24Fc or control IgG Fc was added to macrophages in the last 24 hours.Data shown in FIG. 4A are means and S.D. from three independentexperiments, while those in FIG. 4B are representative of at least 3independent experiments.

FIG. 8 shows a plot of mean plasma CD24Fc concentration (±SD) bytreatment for a PK Evaluable Population in human subjects.PK=pharmacokinetic; SD=standard deviation.

FIG. 9 shows a dose proportionality plot of CD24Fc C_(max) versus dosefor a PK Evaluable Population.

FIG. 10 shows a dose proportionality plot of CD24Fc AUC_(0-42 d) versusdose for a PK Evaluable Population.

FIG. 11 shows a dose proportionality plot of CD24Fc AUC_(0-inf) versusdose for a PK Evaluable Population.

FIG. 12 shows the trial design for the randomized, placebo-controlledPhase IIa dose escalation trial was performed to evaluate the additionof CD24Fc to standard of care acute GVHD prophylaxis in cancer patientsundergoing allogeneic myeloablative hematopoietic stem celltransplantation (HCT).

FIG. 13 shows the dosing scheme for the single dose and multi-dosecohorts in the Phase IIa trial.

FIG. 14 shows the median time to engraftment for patients enrolled inthe trial.

FIG. 15 shows the myeloid donor chimerism for patients enrolled in thetrial.

FIG. 16 shows the incidence of Grade II-IV and Grade III-IV acute GVHDin the treatment (CD24Fc) cohort.

FIG. 17 shows the cumulative incidence of grade III-IV aGVHD 180 dayspost HCT in patients receiving methotrexate/tacrolimus+CD24Fc ascompared to contemporary control patients receivingmethotrexate/tacrolimus.

FIG. 18 shows the Kaplan-Meier survival analysis comparing 180 daysGrade III-IV aGVHD, relapse-free survival in patients receiving eitherCD24Fc or placebo control.

FIG. 19 shows the Kaplan-Meier survival analysis comparing 180 daysGrade III-IV aGVHD, relapse-free survival in patients receiving CD24Fcwith contemporary control.

FIG. 20 shows the Kaplan-Meier survival analysis comparing overallsurvival of patients receiving either CD24Fc or placebo control at 800days post transplant.

FIG. 21 shows the Kaplan-Meier survival analysis comparing overallsurvival of patients receiving CD24Fc or contemporary control at 800days post transplant.

FIGS. 22A-B show the PK data from the 240 and 480 mg single dosecohorts. FIG. 22A Plot of Mean (±Standard Deviation) Plasma CD24FcConcentration (ng/mL) Versus Time on a Linear Scale. FIG. 22B. Plot ofMean (±Standard Deviation) Plasma CD24Fc Concentration (ng/mL) VersusTime on a Semi-Logarithmic Scale.

FIGS. 23A-B show the PK data from the multi-dose cohort. FIG. 23A Plotof Mean (±Standard Deviation) Plasma CD24Fc Concentration (ng/mL) VersusTime on a Linear Scale. FIG. 23B. Plot of Mean (±Standard Deviation)Plasma CD24Fc Concentration (ng/mL) Versus Time on a Semi-LogarithmicScale.

FIGS. 24A-B show the effect of CD24Fc treatment on oral mucositis. FIG.24A. Combined mucositis scores. As a measure of the effect on mucositis,we generated a multiple of the number of days patients spent withserious (Grade 3 or 4) mucositis and this is provided in the bars, alongwith the number of pts that had the mucositis in parentheses. FIG. 24B.Dose-dependent reduction of mucositis score by CD24Fc. Means andstandard errors of individual mucositis score are shown. Correlationcoefficient and P-value between drug dose and mucositis score ofpatients were determined by using Pearson method. R=−0.9983, P=0.0009.

FIGS. 25A-B show the 180 Day Grade III-IV GVHD Free Survival in theCD24Fc group compared to the placebo control group (FIG. 25A) and thecontemporary control group (FIG. 25B).

FIGS. 26A-B show the Relapse Free Survival in the CD24Fc group comparedto the placebo control group (FIG. 26A) and the contemporary controlgroup (FIG. 26B).

DETAILED DESCRIPTION

Tissue damage can lead to the release of proinflammatory cytokines (suchas TNF-α, IL-1β and IL-6), and also the release of damage-associatedmolecular patterns (DAMPs) and pathogen-associated molecular patterns(PAMPs). Both DAMPs and PAMPs can activate antigen-presenting cells(APCs), such as dendritic cells (DCs), by binding to pattern recognitionreceptors (PRRs). The host APCs subsequently activate donor T cells andan immunologic cascade that results in the release of pro-inflammatorycytokines and expansion of the antigen specific allo-reactive T cellsthat target host tissues. It is these events that lead to thedevelopment of GVHD and exacerbate the effects of mucositis. Forexample, RIOM starts as an acute inflammation of oral mucosa, tongue andpharynx following radiotherapy, which coincides with recruitment ofvarious inflammatory cells and release of inflammatory cytokines,chemotactic mediators, and growth factors.

The involvement of tissue damage in mucositis and GVHD raised theprospect that negatively regulating host response to DAMPs by CD24Fc canbe explored for GVHD therapy. The inventors' preclinical studies havedemonstrated that CD24Fc specifically targets DAMP-mediated inflammationand prevents GVHD in mouse models, including a humanized mouse model.Importantly, the drug has advantages over conventional immunosuppressantas it does not cause general immune suppression and use of high doses ofCD24Fc does not block antibody response in non-human primates. The dataalso demonstrate that CD24Fc prevents GVHD but preserves the graftversus leukemia (GVL) effect, making it an ideal drug for prophylaxis ofGVHD in leukemia patients. Finally, the inventors' studies in non-humanprimate demonstrate that CD24Fc does not suppress antigen-specificimmune responses, which suggest that CD24Fc will not likely increaserisk of infection.

The inventors have discovered that a soluble form of CD24 is highlyeffective for preventing Graft versus Host Disease (GVHD) and associatedconditions such as mucositis, as well as for preventing leukemia relapsefollowing HCT. The inventors have also discovered that CD24Fc produced adose-dependent reduction in severe mucositis (grade≥3) among patientsreceiving HCT therapy. These effects may be mediated through DAMPs.Pattern recognition is involved in inflammatory response triggered byboth PAMPs and DAMPs. The inventors have realized that recent studieshave demonstrated that an exacerbated host response to DAMPs may play apart in the pathogenesis of inflammatory and autoimmune disease. DAMPswere found to promote the production of inflammatory cytokines andautoimmune diseases and in animal models, and inhibitors of DAMPs suchas HMGB1 and HSP90 were consequently found to ameliorate rheumatoidarthritis (RA). TLRs, RAGE-R, DNGR (encoded by Clec9A), and Mincle havebeen shown to be receptors responsible for mediating inflammationinitiated by a variety of DAMPs.

The inventors' recent work demonstrated that CD24-Siglec G interactionsdiscriminate innate immunity to DAMPs from PAMPs. Siglec proteins aremembrane-associated immunoglobulin (Ig) superfamily members thatrecognize a variety of sialic acid-containing structures. Most Siglecshave an intra-cellular immune-tyrosine inhibitory motif (ITIM) thatassociates with SHP-1, -2 and Cbl-b to control key regulators ofinflammatory responses. The inventors have reported CD24 as the firstnatural ligand for a Siglec, Siglec G in mouse and Siglec 10 in human.Siglec G interacts with sialylated CD24 to suppress the TLR-mediatedhost response to DAMPs, such as HMGB1, via a SHP-1/2 signalingmechanism.

Human CD24 is a small GPI-anchored molecule encoded by an open-readingframe of 240 base pairs in the CD24 gene. Of the 80 amino acids, thefirst 26 constitute the signal peptide, while the last 23 serve as asignal for cleavage to allow for the attachment of the GPI tail. As aresult, the mature human CD24 molecule has only 31 amino acids. One ofthe 31 amino acids is polymorphic among the human population. A C to Ttransition at nucleotide 170 of the open-reading frame results in thesubstitution of Alanine (A) with Valine (V) at residue 31 of the matureprotein. Since this residue is immediately N-terminal to the cleavagesite, and since the replacement is nonconservative, these two allelesmay be expressed at different efficiencies on the cell surface. Indeed,transfection studies with cDNA demonstrated that the CD24^(v) allele ismore efficiently expressed on the cell surface. Consistent with this,CD24^(v/v) PBL expressed higher levels of CD24, especially on T cells.

The inventors have demonstrated that CD24 negatively regulates hostresponse to cellular DAMPs that are released as a result of tissue ororgan damage, and at least two overlapping mechanisms may explain thisactivity. First, CD24 binds to several DAMPs, including HSP70, HSP90,HMGB1 and nucleolin and represses host response to these DAMPs. To dothis, it is presumed that CD24 may trap the inflammatory stimuli toprevent interaction with their receptors, TLR or RAGE. Second, using anacetaminophen-induced mouse model of liver necrosis and ensuringinflammation, the inventors demonstrated that through interaction withits receptor, Siglec G, CD24 provides a powerful negative regulation forhost response to tissue injuries. To achieve this activity, CD24 maybind and stimulate signaling by Siglec G wherein Siglec G-associatedSHP1 triggers the negative regulation. Both mechanisms may act inconcert as mice with targeted mutation of either gene mounted muchstronger inflammatory response. In fact, DC cultured from bone marrowfrom either CD24^(-/-) or Siglec G^(-/-) mice produced higher levels ofinflammatory cytokines when stimulated with either HMGB1, HSP70, orHSP90. To the inventors' knowledge, CD24 is the only inhibitory DAMPreceptor capable of shutting down inflammation triggered by DAMPs and nodrug is currently available that specifically targets host inflammatoryresponse to tissue injuries. Furthermore, the inventors havedemonstrated the ability of exogenous soluble CD24 protein to alleviateDAMP-mediated autoimmune disease using mouse models of RA, MS and GvHD.

1. Definitions

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting. As used in thespecification and the appended claims, the singular forms “a,” “an” and“the” include plural referents unless the context clearly dictatesotherwise.

For recitation of numeric ranges herein, each intervening number therebetween with the same degree of precision is explicitly contemplated.For example, for the range of 6-9, the numbers 7 and 8 are contemplatedin addition to 6 and 9, and for the range 6.0-7.0, the numbers 6.0, 6.1,6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitlycontemplated.

A “peptide” or “polypeptide” is a linked sequence of amino acids and maybe natural, synthetic, or a modification or combination of natural andsynthetic.

“Substantially identical” may mean that a first and second amino acidsequence are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, or 99% over a region of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150,160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or300 amino acids.

“Treatment” or “treating,” when referring to protection of an animalfrom a disease, means preventing, suppressing, repressing, or completelyeliminating the disease. Preventing the disease involves administering acomposition of the present invention to an animal prior to onset of thedisease. Suppressing the disease involves administering a composition ofthe present invention to an animal after induction of the disease butbefore its clinical appearance. Repressing the disease involvesadministering a composition of the present invention to an animal afterclinical appearance of the disease.

A “variant” may mean a peptide or polypeptide that differs in amino acidsequence by the insertion, deletion, or conservative substitution ofamino acids, but retain at least one biological activity. Representativeexamples of “biological activity” include the ability to bind to atoll-like receptor and to be bound by a specific antibody. Variant mayalso mean a protein with an amino acid sequence that is substantiallyidentical to a referenced protein with an amino acid sequence thatretains at least one biological activity. A conservative substitution ofan amino acid, i.e., replacing an amino acid with a different amino acidof similar properties (e.g., hydrophilicity, degree and distribution ofcharged regions) is recognized in the art as typically involving a minorchange. These minor changes can be identified, in part, by consideringthe hydropathic index of amino acids, as understood in the art. Kyte etal., J. Mol. Biol. 157:105-132 (1982). The hydropathic index of an aminoacid is based on a consideration of its hydrophobicity and charge. It isknown in the art that amino acids of similar hydropathic indexes can besubstituted and still retain protein function. In one aspect, aminoacids having hydropathic indexes of ±2 are substituted. Thehydrophilicity of amino acids can also be used to reveal substitutionsthat would result in proteins retaining biological function. Aconsideration of the hydrophilicity of amino acids in the context of apeptide permits calculation of the greatest local average hydrophilicityof that peptide, a useful measure that has been reported to correlatewell with antigenicity and immunogenicity. U.S. Pat. No. 4,554,101,incorporated fully herein by reference. Substitution of amino acidshaving similar hydrophilicity values can result in peptides retainingbiological activity, for example immunogenicity, as is understood in theart. Substitutions may be performed with amino acids havinghydrophilicity values within ±2 of each other. Both the hydrophobicityindex and the hydrophilicity value of amino acids are influenced by theparticular side chain of that amino acid. Consistent with thatobservation, amino acid substitutions that are compatible withbiological function are understood to depend on the relative similarityof the amino acids, and particularly the side chains of those aminoacids, as revealed by the hydrophobicity, hydrophilicity, charge, size,and other properties.

2. CD24

Provided herein is a CD24 protein, which may comprise a mature CD24 or avariant thereof. Mature CD24 corresponds to the extracellular domain(ECD) of CD24. The mature CD24 may be from a human or another mammal Asdescribed above, mature human CD24 protein is 31 amino acids long andhas a variable alanine (A) or valine (V) residue at its C-terminal end.The mature CD24 protein may comprise the following sequence:

(SEQ ID NO: 1) SETTTGTSSNSSQSTSNSGLAPNPTNATTK(V/A)

The C-terminal valine or alanine may be immunogenic and may be omittedfrom the CD24 protein, which may reduce its immunogenicity. Therefore,the CD24 protein may comprise the amino acid sequence of mature humanCD24 lacking the C-terminal amino acid:

(SEQ ID NO: 2) SETTTGTSSNSSQSTSNSGLAPNPTNATTK

Despite considerable sequence variations in the amino acid sequence ofthe mature CD24 proteins from mouse and human, they are functionallyequivalent, as human CD24Fc has been shown to be active in the mouse.The amino acid sequence of the human CD24 ECD shows some sequenceconservation with the mouse protein (39% identity; Genbank accessionnumber NP_033976). However, it is not that surprising that the percentidentity is not higher as the CD24 ECD is only 27-31 amino acids inlength, depending on the species, and binding to some of itsreceptor(s), such as Siglec 10/G, is mediated by its sialic acid and/orgalactose sugars of the glycoprotein. The amino acid sequence identitybetween the extracellular domains of the human Siglec-10 (GenBankaccession number AF310233) and its murine homolog Siglec-G (GenBankaccession number NP_766488) receptor proteins is 63% (FIG. 2). As aresult of sequence conservation between mouse and human CD24 primarilyin the C-terminus and in the abundance of glycosylation sites,significant variations in the mature CD24 proteins may be tolerated inusing the CD24 protein, especially if those variations do not affect theconserved residues in the C-terminus or do not affect the glycosylationsites from either mouse or human CD24. Therefore, the CD24 protein maycomprise the amino acid sequence of mature murine CD24:

(SEQ ID NO: 3) NQTSVAPFPGNQNISASPNPTNATTRG.

The amino acid sequence of the human CD24 ECD shows more sequenceconservation with the cynomolgus monkey protein (52% identity; UniProtaccession number UniProtKB-I7GKK1) than with mouse. Again, this is notsurprising given that the percent identity is not higher as the ECD isonly 29-31 amino acids in length in these species, and the role of sugarresidues in binding to its receptor(s). The amino acid sequence ofcynomolgous Siglec-10 receptor has not been determined but the aminoacid sequence identity between the human and rhesus monkey Siglec-10(GenBank accession number XP_001116352) proteins is 89%. Therefore, theCD24 protein may also comprise the amino acid sequence of maturecynomolgous (or rhesus) monkey CD24:

(SEQ ID NO: 10) TVTTSAPLSSNSPQNTSTTPNPANTTTKA

The CD24 protein may be soluble. The CD24 protein may further comprisean N-terminal signal peptide, which may allow secretion of the proteinfrom a cell expressing the protein. The signal peptide sequence maycomprise the amino acid sequence MGRAMVARLGLGLLLLALLLPTQIYS (SEQ ID NO:4). Alternatively, the signal sequence may comprise any of those thatare found on other transmembrane or secreted proteins, or those modifiedfrom the existing signal peptides known in the art.

a. Fusion

The CD24 protein may be fused at its N- or C-terminal end to a proteintag, which may comprise a portion of a mammalian Ig protein, which maybe human or mouse or from another species. The portion may comprise anFc region of the Ig protein. The Fc region may comprise at least one ofthe hinge region, CH2, CH3, and CH4 domains of the Ig protein. The Igprotein may be human IgG1, IgG2, IgG3, IgG4, or IgA, and the Fc regionmay comprise the hinge region, and CH2 and CH3 domains of the Ig. The Fcregion may comprise the human immunoglobulin G1 (IgG1) isotype SEQ IDNO: 7. The Ig protein may also be IgM, and the Fc region may comprisethe hinge region and CH2, CH3, and CH4 domains of IgM. The protein tagmay be an affinity tag that aids in the purification of the protein,and/or a solubility-enhancing tag that enhances the solubility andrecovery of functional proteins. The protein tag may also increase thevalency of the CD24 protein. The protein tag may also comprise GST, His,FLAG, Myc, MBP, NusA, thioredoxin (TRX), small ubiquitin-like modifier(SUMO), ubiquitin (Ub), albumin, or a Camelid Ig. Methods for makingfusion proteins and purifying fusion proteins are well known in the art.

Based on preclinical research, for the construction of the fusionprotein CD24Fc identified in the examples, the truncated form of nativeCD24 molecule of 30 amino acids, which lacks the final polymorphic aminoacid before the GPI signal cleavage site (that is, a mature CD24 proteinhaving SEQ ID NO: 2), has been used. The mature human CD24 sequence isfused to a human IgG1 Fc domain (SEQ ID NO: 7). The sequence of the fulllength CD24Fc fusion protein is provided in SEQ ID NO: 5 (FIG. 1A), andthe sequence of the processed version of CD24Fc fusion protein that issecreted from the cell (i.e. lacking the signal sequence which iscleaved off) is provided in SEQ ID NO: 6. Processed polymorphic variantsof mature CD24 (that is, mature CD24 protein having SEQ ID NO: 1) fusedto IgG1 Fc may comprise the amino acid sequence set forth in SEQ ID NO:11 or 12.

b. Production

The CD24 protein may be heavily glycosylated, and may be involved infunctions of CD24 such as costimulation of immune cells and interactionwith a damage-associated molecular pattern molecule (DAMP). The CD24protein may be prepared using a eukaryotic expression system. Theexpression system may entail expression from a vector in mammaliancells, such as Chinese Hamster Ovary (CHO) cells. The system may also bea viral vector, such as a replication-defective retroviral vector thatmay be used to infect eukaryotic cells. The CD24 protein may also beproduced from a stable cell line that expresses the CD24 protein from avector or a portion of a vector that has been integrated into thecellular genome. The stable cell line may express the CD24 protein froman integrated replication-defective retroviral vector. The expressionsystem may be GPEx™.

c. Pharmaceutical Composition

The CD24 protein may be contained in a pharmaceutical composition, whichmay comprise a pharmaceutically acceptable amount of the CD24 protein.The pharmaceutical composition may comprise a pharmaceuticallyacceptable carrier. The pharmaceutical composition may comprise asolvent, which may keep the CD24 protein stable over an extended period.The solvent may be PBS, which may keep the CD24 protein stable for atleast 66 months at −20° C. (−15˜−25° C.). The solvent may be capable ofaccommodating the CD24 protein in combination with another drug.

The pharmaceutical composition may be formulated for parenteraladministration including, but not limited to, by injection or continuousinfusion. Formulations for injection may be in the form of suspensions,solutions, or emulsions in oily or aqueous vehicles, and may containformulation agents including, but not limited to, suspending,stabilizing, and dispersing agents. The composition may also be providedin a powder form for reconstitution with a suitable vehicle including,but not limited to, sterile, pyrogen-free water.

The pharmaceutical composition may also be formulated as a depotpreparation, which may be administered by implantation or byintramuscular injection. The composition may be formulated with suitablepolymeric or hydrophobic materials (as an emulsion in an acceptable oil,for example), ion exchange resins, or as sparingly soluble derivatives(as a sparingly soluble salt, for example). A formulation forsubcutaneous injection may be particularly relevant for an indicationlike lupus and its associated manifestations and complications.

3. Methods of Treatment

a. GVHD

Provided herein is a method of preventing, mitigating or treating Graftversus Host Disease (GVHD) in a subject in need thereof by administeringthe CD24 protein to the subject. The subject may have or be at risk ofdeveloping GVHD. The subject may undergo or may be undergoinghematopoietic stem cell transplantation (HCT). The CD24 protein may beused prophylactically to prevent GVHD in a subject undergoing HCT. TheGVHD may be acute GVHD. The CD24 protein may reduce the subject's riskof grade III-IV acute GVHD. The GVHD may be chronic GVHD, including oralGVHD.

The subject may have a cancer. The cancer may be Acute Myeloid Leukemia(AML), Acute Lymphoblastic Leukemia (ALL), Chronic Myelogenous Leukemia(CML), Myelodysplastic syndrome (MDS), or Chronic MyelomonocyticLeukemia (CMML).

b. Mucositis

Provided herein is a method of preventing, mitigating or treatingmucositis in a subject in need thereof by administering the CD24 proteinto the subject. Also described herein is a method of preventing,mitigating or treating other conditions associated with GVHD or the HCTin a subject in need thereof by administering the CD24 protein to thesubject. The associated condition may be mucositis, which may be oralmucositis. Mucositis is a painful inflammation and ulceration of themucous membranes lining the digestive tract, usually as an adverseeffect of the preconditioning chemotherapy and/or radiotherapy regimenfor HCT. Mucositis can occur anywhere along the gastrointestinal (GI)tract. Oral mucositis refers to the particular inflammation andulceration that occurs in the mouth. Oral mucositis is a common andoften debilitating complication of cancer treatment. Many hematopoieticstem cell transplantation recipients experience mucositis, of which oralmucositis is the most common and most debilitating.

The subject may have or be at risk of developing mucositis, which may beoral mucositis. The subject may undergo or may be undergoing at leastone of chemotherapy and radiation therapy. The CD24 protein may be usedprophylactically to prevent mucositis in a subject. The CD24 protein mayreduce the subject's risk of severe mucositis.

c. Medicaments

Also provided are uses of the CD24 protein in the manufacture of amedicament for uses as described herein.

d. Dose Regimen

The dose of the CD24 protein administered may be 0.01 mg/kg to 1000mg/kg, and may be 1 to 500 mg/kg, depending on the desired effect onGVHD or mucositis and the route of administration. The CD24 protein maybe administered by intravenous (IV) infusion or by subcutaneous,intramural (that is, within the wall of a cavity or organ), orintraperitoneal injection. The dose may be 10-1000 mg, 10-500 mg, 240mg, or 480 mg, which in particular may be suitable where the subject isa human.

The CD24 protein may be administered before or after the stem celltransplant. The CD24 protein may be administered 1-4 days, particularly1 day, before the stem cell transplant. The CD24 protein may also beadministered in multiple doses before or after stem cell transplant. TheCD24 protein may be administered in 2, 3, 4, 5 or 6 bi-weekly doses.Each dose of the CD24 protein may be 240 mg or 480 mg. A first dose maybe administered on day −4 to day 0 relative to the day of stem celltransplant (day 0), and may be administered on day −1 in particular.Each subsequent dose may be administered every 9-19 or 11-17 daysthereafter. A second dose may be administered on day +9 to +19 or day+11 to +17, particularly day +14, relative to the day of stem celltransplant. A third dose may be administered on day +18 to +38, day +23to +33, or day +22 to +34, particularly day +28, relative to the day ofstem cell transplant. In particular, the CD24 protein may beadministered in three biweekly administrations of 480 mg, 240 mg, and240 mg, respectively on day −1, day +14 and day +28 relative to the dayof stem cell transplant. The CD24 protein may in particular be CD24Fc.

The CD24 protein may be administered before or during at least one ofchemotherapy and radiation therapy is administered to the subject. TheCD24 protein may be administered so that it is present when DAMPs arereleased.

e. Combination Treatment

The CD24 protein may be administered to the subject in combination withstandard of care GVHD prophylaxis. The standard of care GVHD prophylaxismay comprise administration of methotrexate plus calcineurin inhibitor,such as tacrolimus (Prograf, FK506) or cyclosporine (Sandimmune,Neoral). Tacrolimus may be administered on day −3 relative to the day ofstem cell transplant, and may be administered by IV or PO (orally). ForIV dosing as a continuous infusion the starting dose may be 0.03mg/kg/day based on adjusted body weight. For oral dosing the startingdose may be 0.045 mg/kg/dose twice daily. If the subject cannot toleratetacrolimus, then cyclosporine may be administered to the subject by IVat a dose of 100× the IV tacrolimus dose (e.g., 3 mg/kg/day startingdose). The cyclosporine may also be administered orally at a dose of 3×the IV dose. When Neoral brand is used, because of greaterbioavailability, the cyclosporine may be administered orally at 2× theIV dose.

In the absence of GVHD, tacrolimus levels may be monitored fortherapeutic dosing only during the first 100 days post-transplant. Thetherapeutic target trough level for tacrolimus may be 5-15 ng/mL.Tacrolimus levels may be monitored at a minimum of three times (e.g.every 48-72 hours) for the first week post CD24 protein infusion (day 0to day 7). In the absence of GVHD or relapse, tacrolimus tapering maybegin on day +100 post-transplant. In the presence of GVHD, tacrolimusmay be continued at the therapeutic dosing.

Methotrexate may be used in combination with tacrolimus for standardGVHD prophylaxis. Methotrexate may be administered intravenously at adose of 15 mg/m²/dose once daily on Day 1 after HCT, and at a dose of 10mg/m²/dose on days 3, 6, and 11 after HCT.

For mucositis, the CD24 protein may be administered with at least oneimmunomodulatory agent (that is, other than a CD24 protein) to minimizethe exacerbating inflammatory component. The CD24 protein may beadministered in combination with allopurinol for subjects treated with5-fluorouracil.

The CD24 protein may be administered in combination with one or more ofthe following myeloablative conditioning regimens, and may beadministered before or during the conditioning regimens. The CD24protein may be administered so that it is present when DAMPs arereleased in response to at least one of a myeloablative conditioningregimen and the HCT.

Busulfan and Fludarabine

Busulfan may be administered on days −5 to −2 relative to the day ofstem cell transplant. The dose may be 3.2 mg/kg/day or 130 mg/m²/day,and may be administered intravenously. The total dose may be 12.8 mg/kgor 520 mg/m². Fludarabine may be administered on days −5 to −2 relativeto the day of stem cell transplant. The dose may be 30-45 mg/m²/day, andthe total dose may be 120-180 mg/m². The sequence and timing of busulfanand fludarabine may be done according to institutional standards formyeoablative conditioning that are known in the art.

Busulfan and Cyclophosphamide

Bulsulfan may be administered on days −7 to −4 relative to the day ofstem cell transplant. The dose may be 3.2 mg/kg/day or 130 mg/m²/day,and may be administered intravenously. The total dose may be 12.8 mg/kgor 520 mg/m². Cyclophosphamide may be administered on days −3 to −2relative to the day of stem cell transplant. The dose may be 60mg/kg/day, and the total dose may be 120 mg/kg.

Cyclophosphamide and Total Body Irradiation

Total body irradiation (TBI) may be administered on days −7 to −4relative to stem cell transplantation. Cyclophosphamide may beadministered on days −3 to −2 relative to the day of stem celltransplant. The dose may be 60 mg/kg/day, and the total dose may be 120mg/kg. The sequence of cyclophosphamide, TBI and TBI administrationpractices for myeloablative regimens may be done according toinstitutional standards for myeoablative conditioning that are known inthe art.

The CD24 protein may also be administered with individual myeloablativetreatments described above or combinations thereof.

EXAMPLE 1 CD24 Pharmacokinetics in Mice

1 mg of CD24Fc (CD24Fc) was injected into naïve C57BL/6 mice andcollected blood samples at different timepoints (5 min, 1 hr, 4 hrs, 24hrs, 48 hrs, 7 days, 14 days and 21 days) with 3 mice in each timepoint.The sera were diluted 1:100 and the levels of CD24Fc was detected usinga sandwich ELISA using purified anti-human CD24 (3.3 μg/ml) as thecapturing antibody and peroxidase conjugated goat anti-human IgG Fc (5μg/ml) as the detecting antibodies. As shown in FIG. 3 a. The decaycurve of CD24Fc revealed a typical biphase decay of the protein. Thefirst biodistribution phase had a half-life of 12.4 hours. The secondphase follows a model of first-order elimination from the centralcompartment. The half-life for the second phase was 9.54 days, which issimilar to that of antibodies in vivo. These data suggest that thefusion protein is very stable in the blood stream. In another study inwhich the fusion protein was injected subcutaneously, an almostidentical half-life of 9.52 days was observed (FIG. 3b ). Moreimportantly, while it took approximately 48 hours for the CD24Fc toreach peak levels in the blood, the total amount of the fusion proteinin the blood, as measured by AUC, was substantially the same by eitherroute of injection. Thus, from a therapeutic point of view, using adifferent route of injection should not affect the therapeutic effect ofthe drug. This observation greatly simplified the experimental designfor primate toxicity and clinical trials.

EXAMPLE 2 CD24-Siglec 10 Interaction in Host Response to Tissue Injuries

Nearly two decades ago, Matzinger proposed what was popularly calleddanger theory. In essence, she argued that the immune system is turnedon when it senses the dangers in the host. Although the nature of dangerwas not well defined at the time, it has been determined that necrosisis associated with the release of intracellular components such as HMGB1and Heat-shock proteins, which were called DAMP, for danger-associatedmolecular patterns. DAMP were found to promote production ofinflammatory cytokines and autoimmune diseases. In animal models,inhibitors of HMGB1 and HSP90 were found to ameliorate RA. Theinvolvement of DAMP raised the prospect that negative regulation forhost response to DAMP can be explored for RA therapy.

Using acetaminophen-induced liver necrosis and ensuring inflammation, itwas observed that through interaction Siglec G, CD24 provides a powerfulnegative regulation for host response to tissue injuries. CD24 is a GPIanchored molecules that is broadly expressed in hematopoietic cells andother tissue stem cells. Genetic analysis of a variety of autoimmunedisease in human, including multiple sclerosis, systemic lupuserythromatosus, RA, and giant cell arthritis, showed significantassociation between CD24 polymorphism and risk of autoimmune diseases.Siglec G is a member of I-lectin family, defined by their ability torecognize sialic acid containing structure. Siglec G recognized sialicacid containing structure on CD24 and negatively regulates production ofinflammatory cytokines by dendritic cells. In terms of its ability tointeract with CD24, human Siglec 10 and mouse Siglec G are functionallyequivalent. However, it is unclear if there is a one-to-one correlationbetween mouse and human homologues. Although the mechanism remains to befully elucidated, it is plausible that Siglec G-associated SHP1 may beinvolved in the negative regulation. These data lead to a new model inwhich CD24-Siglec G/10 interaction may play a critical in discriminationpathogen-associated molecular pattern (PAMP) from DAMP (FIG. 4).

At least two overlapping mechanisms may explain the function of CD24.First, by binding to a variety of DAMP, CD24 may trap the inflammatorystimuli to prevent their interaction with TLR or RAGE. This notion issupported by observations that CD24 is associated with several DAMPmolecules, including HSP70, 90, HMGB1 and nucleolin. Second, perhapsafter associated with DAMP, CD24 may stimulate signaling by Siglec G.Both mechanisms may act in concert as mice with targeted mutation ofeither gene mounted much stronger inflammatory response. In fact, DCcultured from bone marrow from either CD24-/- or Siglec G-/- miceproduced much higher inflammatory cytokines when stimulated with eitherHMGB1, HSP70, or HSP90. In contrast, no effect were found in theirresponse to PAMP, such as LPS and PolyI:C. These data not only provideda mechanism for the innate immune system to distinguish pathogen fromtissue injury, but also suggest that CD24 and Siglec G as potentialtherapeutic targets for diseases associated with tissue injuries.

EXAMPLE 3

CD24Fc Interacts With HMGB1, Siglec 10 and Induces Association BetweenSiglec G and SHP-1

To measure the interaction between CD24Fc and Siglec 10, we immobilizedCD24Fc onto a CHIP and used Biacore to measure the binding of differentconcentrations of Siglec-10Fc. As shown in FIG. 5 a, CD24Fc binds withSiglec 10 with a Kd of 1.6×10⁻⁷M. This is 100-fold higher affinity thanthe control Fc. The interaction between CD24Fc and HMGB1 was confirmedby pull down experiments using CD24Fc-bound protein G beads followed byWestern blot with either anti-IgG or anti-HMGBE These data demonstratethat CD24Fc, but not Fc, binds to HMGB1 and that this binding iscation-dependent (FIG. 5b ). To determine whether CD24Fc is an agonistof Siglec G, the mouse counterpart of human Siglec 10, we stimulatedCD24-/- spleen cells with CD24Fc, control Fc or vehicle (PBS) controlfor 30 minutes. Siglec G was then immunoprecipitated and probed withanti-phospho-tyrosine or anti-SHP-1. As shown in FIG. 5 c, CD24Fcinduced substantial phosphorylation of Siglec G and association ofSHP-1, a well-known inhibitor for both adaptive and innate immunity.

In vitro efficacy studies of CD24Fc.

To study the impact of CD24Fc on the production of inflammatorycytokines by human T cells, the mature T cells in human PBML wereactivated by anti-CD3 antibody (OKT3), a commonly used agonist of the Tcell receptor in the presence of different concentrations of CD24Fc orhuman IgG1 Fc. Four days later, the supernatants were collected and theproduction of IFN-γ and TNF-α were measured by Enzyme-linkedimmunosorbent assay (ELISA) to confirm activation. The results in FIG. 6demonstrated that CD24Fc from two different manufacturing lotssignificantly reduced IFN-γ and TNF-α production from the activatedhuman PBML compared with control IgG Fc control. In addition, whenCD24Fc was added, cytokine production was inhibited in a dose-dependentmanner Therefore, CD24Fc can inhibit anti-CD3 induced human PBMLactivation in vitro. This study not only indicated the mechanism ofaction of CD24Fc might be through the inhibition of T cell activation,but also established a reliable bioassay for drug potency and stabilitytesting.

To determine whether CD24Fc regulates production of inflammatorycytokines in a human cell line, we first silenced CD24 in the humanacute monocytic leukemia THP1 cell line using RNAi, and then induceddifferentiation into macrophages by treating them with PMA. As shown inFIG. 7 a, CD24 silencing substantially increased the production of TNFα,IL-1β and IL-6. These data demonstrate an essential role for endogenoushuman CD24 in limiting the production of inflammatory cytokines.Importantly, CD24Fc restored inhibition of TNFα in the CD24-silencedcell line (FIG. 7b ), as well as IL-1β and IL-6. These data not onlydemonstrate the relevance of CD24 in inflammatory response of humancells, but also provides a simple assay to assess biological activity ofCD24Fc.

Taken together, these data demonstrate that CD24Fc is capable ofinhibiting cytokine production triggered by adaptive and innate stimuli.However, since the drug is much more effective in reducing cytokineproduction by innate effectors, we consider that the primary mechanismfor its prophylactic function is to prevent inflammation triggered bytissue injuries at the early phase of transplantation.

EXAMPLE 4 CD24 Pharmacokinetics in Humans

This example shows an analysis of the pharmacokinetics of a CD24 proteinin humans. This was derived from a Phase I, randomized, double-blind,placebo-controlled, single ascending dose study to assess the safety,tolerability, and PK of CD24Fc in healthy male and female adultsubjects. A total of 40 subjects in 5 cohorts of 8 subjects each wereenrolled in this study. Six of the 8 subjects in each cohort receivedstudy drug and 2 subjects received placebo (0.9% sodium chloride,saline). The first cohort was dosed with 10 mg. Succeeding cohortsreceived 30 mg, 60 mg, 120 mg, and 240 mg of CD24Fc or matching placeboand were dosed at least 3 weeks apart to allow for review of safety andtolerability data for each prior cohort. Administration of the nexthigher dose to a new cohort of subjects was permitted only if adequatesafety and tolerability had been demonstrated.

In each cohort, the initial 2 subjects were 1 study drug recipient and 1placebo recipient on Day 1. The 3rd to 5th and 6th to 8th subjects weredosed after Day 7 (a minimum of 24 hours apart between the subgroups).Each subject was dosed at least 1 hour apart in the same subgroup. Ifnecessary, dosing of the rest of subjects was delayed pending review ofany significant safety issues that may have arisen during the post-doseperiod involving the first or second subgroups in that cohort. Thesubsequent cohort was dosed at least 3 weeks after the prior cohort.

Screening Period:

The Screening Visit (Visit 1) occurred up to 21 days prior to thebeginning of the active treatment period. After providing informedconsent, subjects underwent screening procedures for eligibility.

Treatment Period:

Subjects were admitted to the Clinical Pharmacology Unit (CPU) on Day −1(Visit 2), and the randomized treatment period began on Day 1 followinga 10-hour minimum overnight fast. Subjects were randomly assigned totreatment with CD24Fc or placebo as a single dose. Subjects remainedconfined until the morning of Day 4.

Follow-Up:

All subjects returned to the CPU on Day 7, Day 14, Day 21, Day 28, andDay 42 (±1 day) for follow-up visits (Visit 3, Visit 4, Visit 5, Visit6, and Visit 7). Visit 7 was the final visit for all subjects.

Duration of Treatment:

The total study duration for each subject was up to 63 days. Single-doseadministration occurred on Day 1.

Number of Subjects:

Planned: 40 subjects

Screened: 224 subjects

Randomized: 40 subjects

Completed: 39 subjects

Discontinued: 1 subject

Diagnosis and Main Criteria for Inclusion:

The population for this study was healthy males and females between theages of 18 and 55 years, inclusive, with a body mass index between 18kg/m² and 30 kg/m², inclusive.

Investigational Product and Comparator Information:

CD24Fc: single dose of 10 mg, 30 mg, 60 mg, 120 mg, or 240 mgadministered via IV infusion; lot number: 09MM-036. CD24Fc was a fullyhumanized fusion protein consisting of the mature sequence of human CD24and the fragment crystallizable region of human immunoglobulin G1(IgG1Fc). CD24Fc was supplied as a sterile, clear, colorless,preservative-free, aqueous solution for IV administration. CD24Fc wasformulated as single dose injection solution, at a concentration of 10mg/mL and a pH of 7.2. Each CD24Fc vial contained 160 mg of CD24Fc, 5.3mg of sodium chloride, 32.6 mg of sodium phosphate dibasic heptahydrate,and 140 mg of sodium phosphate monobasic monohydrate in 16 mL±0.2 mL ofCD24Fc. CD24Fc was supplied in clear borosilicate glass vials withchlorobutyl rubber stoppers and aluminum flip-off seals.

Matching placebo (0.9% sodium chloride, saline) administered via IVinfusion; lot numbers: P296855, P311852, P300715, P315952.

The intent-to-treat (ITT) Population consisted of all subjects whoreceived at least 1 dose of the study drug. The ITT Population was theprimary analysis population for subject information and safetyevaluation.

Clinical laboratory evaluations (chemistry, hematology, and urinalysis)were summarized by treatment and visit. Change from baseline was alsosummarized. Vital signs (blood pressure, heart rate, respiratory rate,and temperature) were summarized by treatment and time point. Changefrom baseline was also summarized. All physical examination data werelisted. Electrocardiogram parameters and the change from baseline weresummarized. Overall interpretations were listed.

Plasma CD24Fc Concentration

As shown in FIG. 8, the mean plasma concentration of CD24Fc increasedproportionally to the dose of CD24Fc administered. For all dose groupsexcept 120 mg, the maximum mean plasma concentration of CD24Fc wasreached at 1 hour post-dose. The maximum mean plasma concentration ofCD24Fc for the 120 mg group was reached at 2 hours post-dose. By Day 42(984 hours), the mean plasma concentration of CD24Fc for all groups haddecreased to between 2% and 4% of the maximum mean plasma concentration.

Table 1 summarizes the plasma CD24Fc PK parameters by treatment for thePK Evaluable Population.

TABLE 1 Summary of Plasma CD24Fc Pharmacokinetic Parameters byTreatment - PK Evaluable Population CD24Fc CD24Fc CD24Fc CD24Fc CD24Fc10 mg 30 mg 60 mg 120 mg 240 mg Parameter Statistic (N = 6) (N = 6) (N =6) (N = 6) (N = 6) C_(max) (ng/mL) n 6 6 6 6 6 Mean (SD) 2495 (576) 9735(1715) 30 083 (7179) 52 435 (9910) 95 865 (10 734) CV % 23.1 17.6 23.918.9 11.2 Median 2371 9218   29 026   50 401   93 206 Min, Max 1,967,3,390 8,583, 13,086 22,557, 42,628 40,434, 65,704 81,296, 110,110Geometric mean 2,442 9,625 29,424 51,666 95,365 Geometric CV % 22.8 16.123.0 19.0 11.2 AUC_(0-42 d) (ng*hr/mL) n 6 6 6 6 6 Mean (SD) 423,061(99,615) 1,282,430 (88,798) 3,226,255 (702,862) 6,541,501 (2,190,944)12,704,705 (1,918,596) CV % 23.5 6.9 21.8 33.5 15.1 Median 434,0431,302,719 3,124,933 5,785,142 12,563,426 Min, Max 291,020, 528,0791,175,733, 1,403,024 2,487,550, 4,139,748 4,485,193, 9,415,26610,466,635, 15,693,606 Geometric mean 412,795 1,279,851 3,163,2526,249,552 12,586,731 Geometric CV % 25.0 7.0 22.0 33.8 15.0 AUC_(0-inf)(ng*hr/mL) n 6 6 6 6 6 Mean (SD) 462,260 (116,040) 1,434,464 (131,316)3,497,196 (705,653) 7,198,196 (2,458,320) 13,861,796 (1,962,780) CV %25.1 9.2 20.2 34.2 14.2 Median 470,426 1,422,205 3,519,732 6,463,66513,713,034 Min, Max 310,956, 596,599 1,281,715, 1,650,503 2,703,655,4,309,023 4,910,640, 10,479,940 11,822,988, 17,175,236 Geometric mean449,583 1,429,578 3,437,036 6,862,129 13,750,972 Geometric CV % 26.7 9.020.7 34.6 13.8 T_(max) (hr) n 6 6 6 6 6 Mean (SD) 1.15 (0.42) 1.17(0.41) 1.01 (0.01) 1.34 (0.51) 1.33 (0.52) CV % 36.1 35.0 1.2 38.0 38.7Median 1.00 1.00 1.00 1.03 1.00 Min, Max 0.92, 2.00 1.00, 2.00 1.00,1.03 1.00, 2.00 1.00, 2.00 t_(1/2) (hr) n 6 6 6 6 6 Mean (SD) 280.83(22.37) 327.10 (41.32) 279.82 (65.59) 286.45 (23.38) 285.33 (24.33) CV %8.0 12.6 23.4 8.2 8.5 Median 279.61 317.23 264.69 290.76 287.74 Min, Max258.87, 321.26 289.82, 394.24 210.18, 362.46 243.89, 309.26 249.24,322.26 AUCextr (%) n 6 6 6 6 6 Mean (SD) 7.61 (2.14) 10.44 (2.94) 7.88(4.26) 8.92 (1.94) 8.46 (1.99) CV % 28.1 28.2 54.0 21.8 23.5 Median 7.1610.01 6.35 9.27 8.45 Min, Max 5.46, 11.47 7.10, 15.05 3.92, 14.48 5.49,10.99 5.56, 11.50 CL (L/hr) n 6 6 6 6 6 Mean (SD) 0.0229 (0.0061) 0.0211(0.0019) 0.0178 (0.0036) 0.0183 (0.0058) 0.0176 (0.0023) CV % 26.7 8.820.5 31.7 13.3 Median 0.0216 0.0211 0.0173 0.0191 0.0175 Min, Max0.0168, 0.0322 0.0182, 0.0234 0.0139, 0.0222 0.0115, 0.0244 0.0140,0.0203 Vd (L) n 6 6 6 6 6 Mean (SD) 9.153 (1.943) 9.867 (0.804) 7.289(2.592) 7.491 (2.202) 7.276 (1.426) CV % 21.2 8.1 35.6 29.4 19.6 Median8.507 10.007 7.486 7.691 7.151 Min, Max 7.326, 12.010 8.771, 10.9584.222, 11.139 4.933, 9.974 5.814, 9.438 AUC_(0-42 d) = area under theconcentration-time curve from time 0 to 42 days; AUC_(0-inf) = areaunder the concentration-time curve extrapolated from time 0 to infinity;AUC_(extr) = percentage of AUC_(0-inf) that was due to extrapolationfrom the time of the last measurable concentration, per subject, toinfinity; CL = total body clearance; C_(max) = maximum observed plasmadrug concentration; CV % = coefficient of variation; Min = minimum; Max= maximum; SD = standard deviation; t_(1/2) = terminal eliminationhalf-life; T_(max) = time of maximum observed plasma drug concentration;V_(d) = volume of distribution.

Plasma CD24Fc Dose Proportionality Analysis

FIG. 9 shows a dose proportionality plot of CD24Fc C_(max) versus dosefor the PK Evaluable Population. FIG. 10 shows a dose proportionalityplot of CD24Fc AUC_(0-42 d) versus dose for the PK Evaluable Population.FIG. 11 shows a dose proportionality plot of CD24Fc AUC_(0-inf) versusdose for the PK Evaluable Population. Table 2 shows a power analysis ofdose proportionality.

TABLE 2 Power Analysis of Dose Proportionality: Plasma CD24FcPharmacokinetic Parameters - PK Evaluable Population CD24Fc CD24FcCD24Fc CD24Fc CD24Fc Dose Proportionality Parameter 10 mg 30 mg 60 mg120 mg 240 mg Slope Standard Statistic (N = 6) (N = 6) (N = 6) (N = 6)(N = 6) Estimate Error 90% CI C_(max) (ng/mL) 1.172 0.040 (1.105, 1.240)Geometric mean 2,441.8 9,624.9 29,424.4 51,666.4 95,364.9 Geometric CV %22.8 16.1 23.0 19.0 11.2 AUC_(0-42 d) (ng*hr/mL) 1.088 0.036 (1.027,1.148) Geometric mean 412,794.8 1,279,850.8 3,163,251.7 6,249,551.912,586,731.3 Geometric CV % 25.0 7.0 22.0 33.8 15.0 AUC_(0-inf)(ng*hr/mL) 1.087 0.036 (1.026, 1.148) Geometric mean 449,583.51,429,577.5 3,437,035.6 6,862,128.7 13,750,972.4 Geometric CV % 26.7 9.020.7 34.6 13.8 Geometric CV % = 100*sqrt(exp(SD²) − 1), where SD was thestandard deviation of the log-transformed data. The power model wasfitted by restricted maximum likelihood, regressing the log-transformedPK parameter on log transformed dose. Both the intercept and slope werefitted as fixed effects. Dose proportionality was not rejected if the90% CI lies within (0.8, 1.25). AUC_(0-42 d) = area under theconcentration-time curve from time 0 to 42 days; AUC_(0-inf) = areaunder the concentration-time curve extrapolated from time 0 to infinity;CI = confidence interval; C_(max) = maximum observed plasma drugconcentration; CV % = coefficient of variation; PK = pharmacokinetic; SD= standard deviation.

The C_(max) slope estimate was 1.172 with a 90% CI of 1.105 to 1.240.The AUC_(0-42 d) slope estimate was 1.088 with a 90% CI of 1.027 to1.148. The AUC_(0-inf) slope estimate was 1.087 with a 90% CI of 1.026to 1.1.

Pharmacokinetic Conclusions

The C_(max) and AUCs of plasma CD24Fc increased proportionally to thedoses administered in mouse, monkey and human. The plasma CD24Fc reachedT_(max) between 1.01 and 1.34 hours. The t_(1/2) of plasma CD24Fc rangedbetween 280.83 and 327.10 hours.

EXAMPLE 5 CD24 can be Used to Treat Graft Versus Host Disease in HumanSubjects

A multicenter, prospective, double-blind, randomized, placebo-controlledPhase IIa dose escalation trial was performed to evaluate the additionof a CD24 protein, CD24Fc, to standard of care acute GVHD prophylaxis incancer patients undergoing allogeneic myeloablative hematopoietic stemcell transplantation (HCT). The trial design is shown in FIG. 12.

The primary objectives of the phase IIa study include assessing thesafety and tolerability of CD24Fc in combination with methotrexate andtacrolimus prophylaxis in patients undergoing matched unrelated donorHCT following myeloablative conditioning, and to define the recommendedphase 2 dose (RP2D) or maximum tolerated dose (MTD). In addition,secondary efficacy objectives in the phase IIa study include:

-   -   determining if the addition of CD24Fc to standard GVHD        prophylaxis methotrexate and tacrolimus reduces the cumulative        incidence of grade II-IV aGVHD at day 100 after HCT    -   estimating grade II-IV aGVHD free survival (GFS) at day 180        after HCT,    -   describing the incidence of cGVHD (cGVHD) at 1 year    -   describing the incidence of relapse one year following HCT    -   describing the incidence of transplant-related mortality (TRM)        one year following HCT    -   describing infection rates at day 100 following HCT    -   evaluating overall survival (OS), absence of grade III-IV GVHD,        and relapse-free survival one year following HCT    -   evaluating conditioning toxicity including oral mucositis and        organ failure

Other objectives include assessing the pharmacokinetic (PK) profile ofCD24Fc, examining the immune cell profile and functional responses ofAPCs and T cells after HCT in the CD24Fc and placebo groups, andassessing pharmacodynamics (PD) biomarkers such as the plasmaconcentrations of pro-inflammatory cytokines, DAMPs, lipids, and GVHDbiomarkers in the CD24Fc and placebo groups.

The trial enrolled patients receiving transplants from matched unrelateddonors undergoing allogeneic HCT according to institutional practice.Patients between the ages of 18-70 years old undergoing matchedunrelated donor allogeneic HCT for a malignant hematologic conditionwith a Karnofsky performance score≥70% were eligible for the study. An8/8 HLA allelic match between the unrelated donor and the recipient atHLA-A, HLA-B, HLA-C, and HLA-DRB1 was required. Restricting the study topatients receiving HCT from unrelated donors is expected to limitheterogeneity and facilitate statistical estimates of aGVHD incidencefor subsequent efficacy assessments, given the greater incidence ofgrade II-IV aGVHD (60-80%) and grade III-IV aGVHD (20-35%) in thispopulation.

This trial exclusively utilized myeloablative conditioning regimens andstandard of care (SOC) prophylaxis comprising tacrolimus andmethotrexate since these patients experience the most severe tissueinjury and drug will likely have the strongest biological effect in thissetting. All patients received myeloablative conditioning and standardof care GVHD prophylaxis with methotrexate and tacrolimus per the phaseIIa protocol. Patients received a myeloablative conditioning regimenconsisting of either fludarabine and busulfan (Flu/Bu 4) orcyclophosphamide and total body irradiation (Cy/TBI), as decided by thetreating physician, followed by an infusion of stem cells on day 0. GVHDprophylaxis was administered to all patients and consisted of tacrolimus(initiated Day −3 before transplant) and methotrexate (initiated Day +1after transplant) in combination with CD24Fc in the treatment arm orsaline in the placebo arm. In the absence of GVHD, tacrolimus taperingstarted on day +100. The source of donor stem cells was eitherperipheral blood stem cells (PBSC) or bone marrow (BM).

The Phase IIa trial comprised two single ascending dose cohorts (240 mgand 480 mg) and a single multi-dose cohort of CD24Fc in addition to SOCGVHD prophylaxis as outlined in Table 3 below. As shown in FIG. 13, inthe single dose cohort, the study agent, CD24Fc, was administeredintravenously on day −1 relative to the day of stem cell transplant. Inthe multi-dosing cohort, patients received 3 biweekly administrations ofCD24Fc at 480 mg (day −1), 240 mg (day +14) and 240 mg (day +28). Basedupon PK data for CD24Fc this biweekly dosing period will allow forpassage of greater than two half-lives. Dosing is based on a fixedamount and not based on weight or BSA. Each dosing cohort enrolled 8subjects using a randomized 3:1 ratio (6 CD24Fc subjects and 2 placebo)design for a total enrollment of 24 patients.

Table 4 lists demography information and clinical characteristics forpatients in the CD24Fc and placebo cohorts, which were relativelybalanced across risk factors such as age, malignancy, and comorbidity.The most common malignancy in both the CD24Fc and placebo cohorts wasAML/MDS (66.7% and 83.3%). 72% of the patients in the CD24Fc cohort and50% in the placebo group had a comorbidity index of intermediate orhigh. PBSCs were more frequently used as the graft source as compared tobone marrow in both cohorts, and Flu/Bu 4 was the most commonconditioning regimen across both cohorts. Four patients, all in theCD24Fc cohorts, underwent Cy/TBI conditioning.

TABLE 4 Phase 2a Patient Characteristics CD24Fc + Placebo + TAC/MTXTAC/MTX (N = 18) (N = 6) Age (years) Median (range) 62 (23-68) 57(36-66) Gender (N, %) Female 7 (38.8) 2 (33.3) Male 11 (61.1) 4 (66.7)Graft Source PBSC 15 (83.3) 4 (66.7) (N, %) BM 3 (16.7) 2 (33.3)Malignancy AML/MDS 12 (66.7) 5 (83.3) (N, %) CML 2 (11.1) 0 (0) CMML 1(5.6) 1 (16.7) ALL 3 (16.7) 0 (0) Comorbidity Low (0) 5 3 index (score)Intermediate (1-2) 9 2 High (3-4) 4 1 Cytomegalovirus D+, R+ 5 1 statusD+, R− 1 0 D−, R+ 3 1 D−, R− 8 4 Conditioning Flu/Bu 4 14 (77.8) 6 (100)regimen (N) Cy/TBI 4 (22.2) 0 (0) Engraftment Neutrophil 13.0 (12, 23)15.5 (12, 18) Day (Day) (min, max) Platelets 13.0 (9, 23) 15.0 (11, >48)(min, max) BM = Bone marrow; Cy/TBI = cyclophosphamide/total bodyirradiation; D = donor; Flu/Bu 4 = fludarabine/busulfan; R = recipient

The primary objectives of the study are: to evaluate the safety andtolerability of CD24Fc in subjects undergoing myeloablative allogeneichematopoietic cell transplantation (HCT); and to determine therecommended Phase II dose (RP2D) or maximum tolerable dose (MTD) ofCD24Fc in patients undergoing HCT.

All patients enrolled in the study have completed the Treatment period,which is the first day of treatment with CD24Fc until 30 days after HCTfor the single-dosing cohorts or 60 days after HCT for the multi-dosingcohort (the exact days may vary depending on the last day ofadministration of study drug without constituting a deviation) and isthe assessment and reporting period for adverse events (AE) includingdose limiting toxicities potentially related to the study drug. Table 5provides a summary of toxicities observed in the Phase 2a trial. Overallthis study demonstrated that IV administration of CD24Fc up to 480 mg isgenerally well tolerated in the intent-to-treat (ITT) population. Noinfusion toxicities, dose-limiting toxicities (DLTs) or SAEsattributable or likely attributable to the study drug have been observedand no patients have been removed from the study.

All 24 subjects enrolled engrafted following transplant as shown in FIG.14. Neutrophils engrafted a median of 13.0 and 15.5 days after HCT inCD24Fc exposed and placebo patients, respectively. Platelets engrafted amedian of 13.0 and 15.0 days after HCT in CD24Fc exposed and placebopatients, respectively, with the exception of one patient in the placebogroup in whom platelets did not engraft and who died on Day 49. Therewere no cases of graft failure. The median CD3 chimerism at day +30 was82.5% (range 38-100%) in the CD24Fc exposed patients and 82.0% (range,62%-91%) in the placebo group (FIG. 15). The median donor CD3 chimerismincreased to 86% (range, 42%-100%) at day 100 in the CD24Fc exposedpatients and 84% (range, 17%-100%) in the placebo group. Donor CD33chimerism at day 30 and 100 was 100% in both the CD24Fc and placebogroups.

TABLE 5 Summary of Toxicities Infusion Cohort Treatment # of Pts Rxn SAEDLT Single CD24Fc 6 0 1 0 240 mg Placebo 2 0 2 0 Single CD24Fc 6 0 0 0480 mg Placebo 2 0 1 0 Multidose CD24Fc 6 0 4 0 Placebo 2 0 2 1

Efficacy analyses for the Phase 2a study are considered secondary andinclude the following: to describe grade III-IV acute GVHD free survival(GFS) at day 180 following HCT; to describe the cumulative incidence ofgrade II-IV acute GVHD at day 100 after HCT; to describe grade III-IVGVHD, Relapse Free Survival at day 180 after HCT; to describe gradeII-IV acute GFS at day 180 following HCT; to describe incidence ofchronic GVHD at one year following HCT; to describe incidence of relapseat one year following HCT; to describe incidence of transplant-relatedmortality (TRM) at one year following HCT; to describe rates ofinfection at day 100 following HCT; to evaluate overall survival (OS)and disease free survival (DFS) at one year following HCT.

In addition to inclusion of the placebo arm in the phase IIa study, dataon contemporary controls (N=92) were collected from the sameinstitutions undergoing matched unrelated donor HCT following the samemyeloablative conditioning and GVHD prophylaxis regimens (minus theexperimental therapy CD24Fc) from the period of January 2012 to November2017. A contemporary control cohort was included given the small numberof patients in the placebo control arm. The demography data of the 92adult patients in the contemporary control cohort is summarized in Table6.

TABLE 6 Characteristics of Patients Enrolled in the Contemporary ControlCohort TAC/MTX (N = 92) Age (years) Median (range) 49 (21-69) GenderFemale 41 (44.5) (N, %) Male 51 (55.5) Malignancy AML/MDS 63 (68.5) (N,%) CML 3 (3) CMML 2 (2) ALL 24 (26.5)

Tables 7 and 8 provide an overview of the clinical outcomes of the Ph 2astudy. Acute GVHD was graded according to consensus guidelines utilizedby the international CIBMTR registry and Blood and Marrow TransplantClinical Trials Network and recorded weekly. Patients were evaluated foraGVHD following receipt of HCT on day 0 until day 100 after HCT.

TABLE 7 Overview of Clinical Outcomes, including the cumulativeincidence of grade II-IV and grade III-IV aGVHD. aGVHD Relapse DeathCohort (Day 180) (Day 180) (Day 180) 1) Single Dose: Gr II: 2 (skin) 0 0240 mg Gr III: 0 (N = 6) Gr IV: 0 Gr II-IV: 33.3% (95% CI, 3.2, 70.4) GrIII-IV: 0% 2) Single Dose: Gr II: 2 (Skin and Upper GI) 1 0 480 mg GrIII: 1 (Lower GI) CMML (d 161)   (N = 6) Gr IV: 0 Gr II-IV: 50.0% (95%CI, 7.7, 82.9) Gr III-IV: 16.7% 3) Multi-Dose: Gr II: 2 1 (N = 6) (Skinand Upper GI) ALL (d 103) 0 Gr III: 0 Gr IV: 0 Gr II-IV: 33.3% (95% CI,3.2, 70.4) Gr III-IV: 0% CD24Fc Total: Gr II: 6  2/18 (11.1%) 0/18* GrIII: 1 Gr IV: 0 Gr II-IV: 38.9% (95% CI, 16.8, 60.7) Gr III-IV: 5.6%Placebo Gr II: 0  2/6 (33.3%)  1/6 (16.7%) (N = 6) Gr III: 1 (Lower GI)Gr IV: 0 Gr II-IV: 16.7% (95% CI, 0.5, 54.9) Gr III-IV: 16.7%Contemporary Control Gr II: 22 19/92 (23.1%) 22/92 (23.9%) (N = 92)Grill: 16 (Death as Gr IV: 3 competing Gr II-IV: 50% factor) (death ascompeting factor) (95% CI, 39.8, 60.2) Gr III-IV: 24% (death ascompeting factor) (95% CI, 17.31) *Deaths (n = 2) after day 180: Cohort1 Pneumonitis 2/2 infections (d 210) and Cohort 2 relapse of CMML (d196)

TABLE 8 Summary of Clinical Outcomes Statistical CD24Fc PlaceboSignificance Number 18 6 aGVHD III-IV D 100  6% 17% D 180  6% 17% 1 yrRelapse 11% 33% 1 yr NRM  6% 17% 1.5 yr RFS 83% 50% 1.5 yr OS 89% 50% P= 0.046 D 180 Gr III-IV, Relapse 83% 33% P = 0.01 Free Survival

Incidence of Grade II to IV Acute Graft-Versus-Host Disease by Day 100

Table 9 summarizes the cumulative incidence of Grade II to IV acute GVHDby Day 100 for the mITT Population. In total, 7 (38.9%) patients whoreceived CD24Fc (2 [33.3%] patients in the 240 mg CD24Fc single dosecohort, 3 [50.0%] patients in the 480 mg CD24Fc single dose cohort, and2 [33.3%] patients in the 960 mg CD24Fc multiple dose cohort) and 1(16.7%) patient who received placebo had Grade II to IV acute GVHD byDay 100. Additionally, 1 (16.7%) patient who received placebo diedwithout Grade II to IV acute GVHD by Day 100. Patients who were alivewith no occurrence of Grade II to IV acute GVHD through Day 100 werecensored at their last assessment for acute GVHD on or prior to Day 100.At least 50.0% of patients in each treatment group were censored.

TABLE 9 Cumulative Incidence of Grade II to IV Acute Graft- Versus-HostDisease by Day 100 - mITT Population CD24Fc CD24Fc CD24Fc 240 mg 480 mg960 mg Single Single Multiple CD24Fc Placebo Dose Dose Doses TotalStatistic (N = 6) (N = 6) (N = 6) (N = 6) (N = 18) No. of patients withGrade II-IV 1 (16.7) 2 (33.3) 3 (50.0) 2 (33.3) 7 (38.9) acute GVHD byDay 100 (n, %) No. of patients who died without 1 (16.7) 0 (0.0)  0(0.0)  0 (0.0)  0 (0.0) Grade II-IV acute GVHD by Day 100 (n, %) No. ofpatients censored (n, %) 4 (66.7) 4 (66.7) 3 (50.0) 4 (66.7) 11 (61.1)Cumulative incidence (%) of 16.7 33.3 50.0 33.3 38.9 Grade II-IV acuteGVHD by Day 100 [1] 95% CI (0.5, 54.9) (3.2, 70.4) (7.7, 82.9) (3.2,70.4) (16.8, 60.7) Treatment comparison; CD24Fc versus placebo [2]Hazard ratio (90% CI) 2.6 (0.5, 14.7) Note: Day 100 = Day 100 (+7 days)post-transplant (ie, Study Day 108). Percentage was calculated using thenumber of patients in the column heading as the denominator. [1] Gradeswere based on the CIBMTR grading scale. The cumulative incidence (%) ofacute GVHD by Day 100 and the 95% CI were estimated using the cumulativeincidence function with death without Grade II to IV acute GVHD as acompeting risk. [2] Hazard ratio and 90% CI were based on a Fine andGray model with treatment as a covariate and death without Grade II toIV acute GVHD as a competing risk. CI = confidence interval; CIBMTR =Center for International Blood and Marrow Transplant Research; GVHD =graft-versus-host disease; No. = number.

Overall, the cumulative incidence of Grade II to IV acute GVHD by Day100 (with 95% CI) was 38.9% (16.8%, 60.7%) for the CD24Fc treatmentgroup and 16.7% (0.5%, 54.9%) for the placebo group. The hazard ratio(with 90% CI) for CD24Fc versus placebo was 2.6 (0.5, 14.7). Thecumulative incidence of grade II-IV aGVHD was 50% in the contemporarycontrol. In the CD24Fc treated group, four cases of grade II aGVHDinvolved skin only and two cases involved skin and the uppergastrointestinal (GI) tract. There were no cases grade II aGVHD in theplacebo group.

Grade II-IV Acute Graft-Versus-Host Disease-Free Survival Through Day180

Table 10 summarizes Grade II to IV acute GFS through Day 180 for themITT Population. The median Grade II to IV acute GFS Kaplan-Meierestimate was not reached in any treatment group. Overall, the Grade IIto IV acute GFS rate at Day 180 (with 95% CI) was 61.1% (35.3%, 79.2%)for the CD24Fc treatment group and 50.0% (11.1%, 80.4%) for the placebogroup. The hazard ratio (with 90% CI) for CD24Fc versus placebo was 0.8(0.3, 2.5). Patients who were alive and had no documented occurrence ofGrade II to IV acute GVHD at the data cutoff date were censored at thelast date of acute GVHD assessment on or prior to Day 180. In additionto the small sample size, at least 50.0% of patients in each treatmentgroup were censored.

TABLE 10 Grade II to IV Acute Graft-Versus-Host Disease-Free SurvivalThrough Day 180 - mITT Population CD24Fc CD24Fc CD24Fc 240 mg 480 mg 960mg Single Single Multiple CD24Fc Placebo Dose Dose Doses Total Statistic(N = 6) (N = 6) (N = 6) (N = 6) (N = 18) No. of patients with events (n,%) 3 (50.0) 2 (33.3) 3 (50.0) 2 (33.3) 7 (38.9) Earliest contributingevent Acute GVHD (Grade II-IV) 2 (33.3) 2 (33.3) 3 (50.0) 2 (33.3) 7(38.9) Death 1 (16.7) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) No. of patientscensored (n, %) 3 (50.0) 4 (66.7) 3 (50.0) 4 (66.7) 11 (61.1) Acute GVHD(Grade II-IV)-free survival (days) Kaplan-Meier estimate [1] Median (95%CI) NE NE NE NE NE Mean (SD) [2] 144.2 (74.98) 145.7 (77.01) 116.2(86.69) 150.5 (68.96) 137.4 (74.81) Median [2] 190.0 195.0 121.0 195.0195.0 Min, max [2] 46, 195+ 32, 195+ 24, 195+ 59, 195+ 24, 195+Treatment comparison: CD24Fc versus placebo [3] Hazard ratio (90% CI)0.8 (0.3, 2.5) Rate (%) of being alive without acute 50.0 (11.1, 80.4)66.7 (19.5, 90.4) 50.0 (11.1, 80.4) 66.7 (19.5, 90.4) 61.1 (35.3, 79.2)GVHD (Grade II-IV) at Day 180 (95% CI) [4] Note: Day 180 = Day 180 (+14days) post-transplant (ie, Study Day 195). Percentage was calculatedusing the number of patients in the column heading as the denominator.[1] The 95% CI for median was computed using the Brookmeyer and Crowleymethod with log-log transformation. [2] Censoring was ignored in thecalculation for mean (SD) and median. A “+” after the min or maxindicates a censored observation. [3] Hazard ratio and 90% CI were basedon a Cox proportional hazards model with treatment as a covariate. [4]Kaplan-Meier estimate. CI = confidence interval; GVHD =graft-versus-host disease; log = logarithm; max = maximum; min =minimum; NE = not estimable; No. = number; SD = standard deviation.

Grade III to IV Acute GFS Through Day 180 for the mITT Population

As shown in Table 11, in total, 1 (5.6%) patient who received CD24Fc (1[16.7%] patient in the 480 mg CD24Fc single dose cohort) and 2 (33.3%)patients who received placebo had Grade III to IV acute GVHD by Day 180.Overall, the Grade III to IV acute GFS rate at Day 180 (with 95% CI) was94.4% (66.6%, 99.2%) for the CD24Fc treatment group and 50.0% (11.1%,80.4%) for the placebo group. The hazard ratio (with 90% CI) for CD24Fcversus placebo was 0.1 (0.0, 0.7). Patients who were alive and had nodocumented occurrence of Grade III to IV acute GVHD at the data cutoffdate were censored at the last date of acute GVHD assessment on or priorto Day 180. At least 50.0% of patients in each treatment group werecensored. Grade III to IV acute GFS rate at Day 180 was 24% in thecontemporary control cohort. FIG. 25 shows the 180 Day Grade III-IV GVHDFree Survival in the CD24Fc group compared to the placebo control group(FIG. 25A) and the contemporary control group (FIG. 25B).

TABLE 11 Grade III to IV Acute Graft-Versus-Host Disease-Free SurvivalThrough Day 180 - mITT Population CD24Fc CD24Fc CD24Fc 240 mg 480 mg 960mg Single Single Multiple CD24Fc Placebo Dose Dose Doses Total Statistic(N = 6) (N = 6) (N = 6) (N = 6) (N = 18) No. of patients with events (n,%) 3 (50.0) 0 (0.0) 1 (16.7) 0 (0.0) 1 (5.6) Earliest contributing eventAcute GVHD (Grade III-IV) 2 (33.3) 0 (0.0) 1 (16.7) 0 (0.0) 1 (5.6)Death 1 (16.7) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) No. of patients censored(n, %) 3 (50.0) 6 (100.0) 5 (83.3) 6 (100.0) 17 (94.4) Acute GVHD (GradeIII-IV)-free survival (days) Kaplan-Meier estimate [1] Median (95% CI)NE NE NE NE NE Mean (SD) [2] 144.2 (74.98) 195.0 (0.00) 166.5 (69.81)195.0 (0.00) 185.5 (40.31) Median [2] 190.0 195.0 195.0 195.0 195.0 Min,max [2] 46, 195+ 195+, 195+ 24, 195+ 195+, 195+ 24, 195+ Treatmentcomparison: CD24Fc versus placebo [3] Hazard ratio (90% CI) 0.1 (0.0,0.7) Rate (%) of being alive without acute 50.0 (11.1, 80.4) 100.0 (NE)83.3 (27.3,97.5) 100.0 (NE) 94.4 (66.6, 99.2) GVHD (Grade III-IV) at Day180 (95% CI) [4] Note: Day 180 = Day 180 (+14 days) post-transplant (ie,Study Day 195). Percentage was calculated using the number of patientsin the column heading as the denominator. [1] The 95% CI for median wascomputed using the Brookmeyer and Crowley method with log-logtransformation. [2] Censoring was ignored in the calculation for mean(SD) and median. A “+” after the min or max indicates a censoredobservation. [3] Hazard ratio and 90% CI were based on a Coxproportional hazards model with treatment as a covariate. [4]Kaplan-Meier estimate. CI = confidence interval; GVHD =graft-versus-host disease; log = logarithm; max = maximum; min =minimum; NE = not estimable; No. = number; SD = standard deviation.

All patients who developed aGVHD in the study at the time of the datacutoff have responded to steroid treatment, as compared to the 50%response rate observed in the contemporary cohort control. After thefirst one hundred days post HCT, patients were evaluated quarterly forlate onset aGVHD (defined as acute GVHD onset after day 100) or cGVHDuntil one year after HCT. No additional aGVHD events were observed inthe CD24Fc cohorts after Day 100 post-transplant.

FIG. 16 shows the cumulative incidence of Grade II-IV and Grade III-IVacute GVHD in the treatment (CD24Fc) cohort. In particular, only onepatient had Grade III GVHD that involved the lower GI and no liver GVHDwas observed. The cumulative incidence of grade III-IV aGVHD at Day 180after HCT in the CD24Fc cohorts trended lower than the grade III-IVaGVHD at Day 180 in the contemporary control (P=0.097). There was anadditional case of Grade III aGVHD at Day 182, which resulted in deathat day 184, in a patient in the placebo group. This patient had aleukemia relapse at Day 145. These results suggest that CD24Fc inaddition to methotrexate and tacrolimus prophylaxis reduces the risk ofdeveloping more serious grade III and IV aGVHD in patients undergoingHCT after receiving myeloablative conditioning.

Disease-Free Survival 1 Year Following Hematopoietic Stem CellTransplantation

Table 12 summarizes disease-free survival (DFS) 1 year post-HCT for themITT Population. The median DFS Kaplan-Meier estimate was not reachedfor any treatment group. Overall, the DFS rate at 1 year post-HCT (with95% CI) was 83.3% (56.8%, 94.3%) for the CD24Fc treatment group and50.0% (11.1%, 80.4%) for the placebo group. The hazard ratio (with 90%CI) for CD24Fc versus placebo was 0.2 (0.1, 0.9). Patients who werealive and did not experience disease relapse at the end of the follow-upperiod were censored at the last date of evaluation. At least 50.0% ofpatients in each treatment group were censored. FIG. 26 shows theRelapse Free Survival in the CD24Fc group compared to the placebocontrol group (FIG. 26A) and the contemporary control group (FIG. 26B).

TABLE 12 Disease-Free Survival 1 Year Following Hematopoietic Stem CellTransplantation - mITT Population CD24Fc CD24Fc CD24Fc 240 mg 480 mg 960mg Single Single Multiple CD24Fc Placebo Dose Dose Doses Total Statistic(N = 6) (N = 6) (N = 6) (N = 6) (N = 18) No. of patients with events (n,%) 3 (50.0) 1 (16.7) 1 (16.7) 1 (16.7) 3 (16.7) Earliest contributingevent Relapse 2 (33.3) 0 (0.0) 1 (16.7) 1 (16.7) 2 (11-1) Death 1 (16.7)1 (16.7) 0 (0.0) 0 (0.0) 1 (5.6) No. of patients censored (n, %) 3(50.0) 5 (83.3) 5 (83.3) 5 (83.3) 15 (83.3) Disease-free survival (days)Kaplan-Meier estimate [1] Median (95% CI) NE NE NE NE NE Mean (SD) [2]232.7 (152.48) 342.5 (64.11) 335.2 (92.82) 324.2 (109.89) 333.9 (85.76)Median [2] 256.0 366.0 370.5 366.0 366.0 Min, max [2] 49, 371+ 212, 378+146, 380+ 100, 376+ 100, 380+ Treatment comparison: CD24Fc versusplacebo [3] Hazard ratio (90% CI) 0.2 (0.1, 0.9) Rate (%) of being alivewithout 50.0 (11.1, 80.4) 83.3 (27.3, 97.5) 83.3 (27.3, 97.5) 83.3(27.3, 97.5) 83.3 (56.8, 94.3) relapse at 1 year post-HCT (95% CI) [4]Note: One year = Day 365 (+14 days) post-transplant (ie, Study Day 380).Percentage was calculated using the number of patients in the columnheading as the denominator. [1] The 95% CI for median was computed usingthe Brookmeyer and Crowley method with log-log transformation. [2]Censoring was ignored in the calculation for mean (SD) and median. A “+”after the min or max indicates a censored observation. [3] Hazard ratioand 90% CI were based on a Cox proportional hazards model with treatmentas a covariate. [4] Kaplan-Meier estimate. For Day 365, if the maximumobserved time was < Study Day 380, the Kaplan-Meier estimate at themaximum observed time is presented for a treatment group. CI =confidence interval; HCT = hematopoietic stem cell transplantation; log= logarithm; max = maximum; min = minimum; NE = not estimable; No. =number; SD = standard deviation.

Overall Survival 1 Year Following Hematopoietic Stem CellTransplantation

Table 13 summarizes overall survival (OS) 1 year post-HCT for the mITTPopulation. The median OS time Kaplan-Meier estimate was not reached forany treatment group. Overall, the OS rate at 1 year (with 95% CI) was83.3% (56.8%, 94.3%) for the CD24Fc treatment group and 50.0% (11.1%,80.4%) for the placebo group. The hazard ratio (with 90% CI) for CD24Fcversus placebo was 0.2 (0.1, 1.0). Patients who were alive at the end ofthe follow-up period were censored at the last date that they were knownto be alive. At least 50.0% of patients in each treatment group werecensored.

TABLE 13 Disease-Free Survival 1 Year Following Hematopoietic Stem CellTransplantation - mITT Population CD24Fc CD24Fc CD24Fc 240 mg 480 mg 960mg Single Single Multiple CD24Fc Placebo Dose Dose Doses Total Statistic(N = 6) (N = 6) (N = 6) (N = 6) (N = 18) No. of patients who died (n, %)3 (50.0) 1 (16.7) 1 (16.7) 1 (16.7) 3 (16.7) No. of patients censored(n, %) 3 (50.0) 5 (83.3) 5 (83.3) 5 (83.3) 15 (83.3) Overall survival(days) Kaplan-Meier estimate [1] Median (95% CI) NE NE NE NE NE Mean(SD) [2] 276.3 (132.25) 343.0 (64.34) 343.5 (72.45) 367.2 (6.01) 351.2(53.91) Median [2] 341.5 366.5 370.5 366.0 366.5 Min, max [2] 49, 371+212, 378+ 196, 380+ 358+, 376+ 196, 380+ Treatment comparison; CD24Fcversus placebo [3] Hazard ratio (90% CI) 0.2 (0.1, 1.0) Rate (%) ofbeing alive at 1 year 50.0 (11.1, 80.4) 83.3 (27.3, 97.5) 83.3 (27.3,97.5) 83.3 (27.3, 97.5) 83.3 (56.8, 94.3) post-HCT (95% CI) [4] Note:One year = Day 365 (+14 days) post-transplant (ie, Study Day 380).Percentage was calculated using the number of patients in the columnheading as the denominator. [1] The 95% CI for median was computed usingthe Brookmeyer and Crowley method with log-log transformation. [2]Censoring was ignored in the calculation for mean (SD) and median. A “+”after the min or max indicates a censored observation. [3] Hazard ratioand 90% CI were based on a Cox proportional hazards model with treatmentas a covariate. [4] Kaplan-Meier estimate. For Day 365, if the maximumobserved time was < Study Day 380, the Kaplan-Meier estimate at themaximum observed time is presented for a treatment group. CI =confidence interval; HCT = hematopoietic stem cell transplantation; log= logarithm; max = maximum; min = minimum; NE = not estimable; No. =number; SD = standard deviation.

Estimates of overall survival (OS) at about 800 days post HCT forpatients in the phase IIa study are also encouraging. Overall survival(OS) was about 80% for patients in the CD24Fc cohorts, 50% for patientsin the placebo cohort (p=0.06) (FIG. 20), and 50% for patients in thecontemporary control (p=0.05) (FIG. 21). The improved OS in the CD24Fcexposed patients as compared to the placebo and contemporary controlpatients supports the other findings described above which show thatadministration of CD24Fc in combination with methotrexate and tacrolimusmay yield a substantial improvement on the outcome in patientsundergoing HCT following myeloablative conditioning.

Acute Graft-Versus-Host Disease-Free Survival and Relapse Free Survival(aGRFS) Through Day 180

Therapeutic strategies designed to prevent GVHD may result in anincrease in leukemia relapse due to a reduction in the Graft VersusLeukemia (GVL) effect. As shown in Table 7, the incidence of leukemiarelapse in patients exposed to CD24Fc at Day 180 post HCT (11%) is loweras compared to patients in the placebo group (33%) and the contemporarycontrol (23%). One subject in the 480 mg CD24Fc cohort experiencedrelapse of CMML on Day 146 and one subject in the multi-dose 960 mgCD24Fc cohort experienced relapse of ALL on Day 100 post HCT. Thepatient with CMML passed away on Day 196 due to leukemia. The patientwith ALL relapse was treated with blinatumomab, achieved completeremission, and was alive as of the data cutoff of Aug. 8, 2018. In theplacebo cohort, one patient experienced relapse of CMML on Day 94 andone patient with MDS relapsed on Day 146 (the patient with CMML passedaway on Day 316 and the patient with MDS passed away on Day 184). Theseresults suggest CD24Fc does not interfere with the beneficialgraft-versus-tumor (GVT) process, and may even reduce the risk ofleukemia relapse.

The number of deaths in the CD24Fc cohorts at Day 180 post transplant islower than in the placebo and contemporary control cohorts (Table 7). AtDay 180 post HCT, there were no deaths in any of the CD24Fc cohorts, onedeath due to pneumonia in the placebo cohort (16.7%), and 22 deaths inthe contemporary control (23.9%). Statistically significant improvementsin the composite endpoint of aGVHD grade III-IV, relapse-free survival(RFS) are observed in the CD24Fc cohorts (83%) as compared to theplacebo group (33%) at Day 180 post HCT (P=0.011, see FIG. 18) and thecontemporary control (53%) at Day 180 post HCT (P=0.017, see FIG. 19).This sort of endpoint has become increasingly popular because it intheory encapsulates the effect of an intervention not only on GVHDsuppression but potential impact toxicity, infection and relapse. Insupport of the observations above, improvements in grade III-IV aGVHD,RFS show that administering CD24Fc in combination with a standard orcare methotrexate and tacrolimus following a myeloablative conditioningregimen is beneficial to patients in preventing the occurrence of aGVHDwhile not affecting the GVL effects of the graft.

The aGRFS through Day 180 post-HCT is a post hoc composite endpoint inwhich events included Grade III to IV acute GVHD, relapse, or death fromany cause. Table 14 summarizes the Grade III to IV acute GRFS throughDay 180 for the mITT Population.

The Kaplan-Meier estimate of the median Grade III to IV acute GRFS wasnot reached for the CD24Fc treatment groups. For the placebo group, theKaplan-Meier estimate of the median Grade III to IV acute GRFS (with 95%CI) was 120.0 (46.0, not estimable). Overall, the Grade III to IV acuteGRFS rate at Day 180 (with 95% CI) was 83.3% (56.8%, 94.3%) for theCD24Fc treatment group and 33.3% (4.6%, 67.6%) for the placebo group.The hazard ratio (with 90% CI) for CD24Fc versus placebo was 0.2 (0.0,0.6). Patients who were alive and had no documented occurrence of GradeIII to IV acute GVHD, chronic GVHD requiring systemic immunosuppressivetherapy, or relapse at the data cutoff date were censored at the lastassessment date.

TABLE 14 Grade III to IV Acute Graft-Versus-Host Disease-Free Survivaland Relapse Free Survival Through Day 180 - mITT Population CD24FcCD24Fc CD24Fc 240 mg 480 mg 960 mg Single Single Multiple CD24Fc PlaceboDose Dose Doses Total Statistic (N = 6) (N = 6) (N = 6) (N = 6) (N =1 8)No. of patients with events (n, %) 4 (66.7) 0 (0.0) 2 (33.3) 1 (16.7) 3(16.7) Earliest contributing event Acute GVHD (Grade III-IV) 1 (16.7) 0(0.0) 1 (16.7) 0 (0.0) 1 (5.6) Relapse 2 (33.3) 0 (0.0) 1 (16.7) 1(16.7) 2 (11.1) Death 1 (16.7) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) No. ofpatients censored (n, %) 2 (33.3) 6 (100.0) 4 (66.7) 5 (83.3) 15 (83.3)Grade III-IV acute GVHD-free survival and relapse-free survival (days)Kaplan-Meier estimate [1] Median (95% CI) 120.0 (46.0, NE) NE NE NE NEMean (SD) [2] 120.8 (67.99) 195.0 (0.00) 158.3 (68.67) 179.2 (38.78)177.5 (45.47) Median [2] 120.0 195.0 195.0 195.0 195.0 Min, max [2] 46,195+ 195+, 195+ 24, 195+ 100, 195+ 24, 195+ Treatment comparison: CD24Fcversus placebo [3] Hazard ratio (90% CI) 0.2 (0.0, 0.6) Rate (%) ofbeing alive without 33.3 (4.6, 67.6) 100.0 (NE) 66.7 (19.5, 90.4) 83.3(27.3, 97.5) 83.3 (56.8, 94.3) Grade III-IV acute GVHD or relapse at Day180 (95% CI) [4] Note: Day 180 = Day 180 (+14 days) post-transplant (ie,Study Day 195). Percentage was calculated using the number of patientsin the column heading as the denominator. [1] The 95% CI for median wascomputed using the Brookmeyer and Crowley method with log-logtransformation. [2] Censoring was ignored in the calculation for mean(SD) and median. A “+” after the min or max indicates a censoredobservation. [3] Hazard ratio and 90% CI were based on a Coxproportional hazards model with treatment as a covariate. [4]Kaplan-Meier estimate. CI = confidence interval; GVHD =graft-versus-host disease; log = logarithm; max = maximum; min =minimum; NE = not estimable; No. = number; SD = standard deviation.

Incidence of Relapse 1 Year Following Hematopoietic Stem CellTransplantation

Table 15 summarizes the cumulative incidence of relapse 1 year post-HCTfor the mITT Population. Overall, the cumulative incidence rate ofrelapse at 1 year post-HCT (with 95% CI) was 11.1% (1.7%, 30.4%) for theCD24Fc treatment group and 33.3% (2.9%, 71.1%) for the placebo group.The hazard ratio (with 90% CI) for CD24Fc versus placebo was 0.3 (0.1,1.4). Patients who were alive and did not experience relapse at the endof the follow-up period (Day 365 [1 year]) were censored at the lastdate of evaluation. At least 50.0% of patients in each treatment groupwere censored.

TABLE 15 Cumulative Incidence of Relapse 1 Year Following HematopoieticStem Cell Transplantation - mITT Population CD24Fc CD24Fc CD24Fc 240 mg480 mg 960 mg Single Single Multiple CD24Fc Placebo Dose Dose DosesTotal Statistic (N = 6) (N = 6) (N = 6) (N = 6) (N = 18) No. of patientswith relapse by 1 year 2 (33.3) 0 (0.0) 1 (16.7) 1 (16.7) 2 (11.1)post-HCT (n, %) No. of patients who died without 1 (16.7) 1 (16.7) 0(0.0) 0 (0.0) 1 (5.6) relapse by 1 year post-HCT (n, %) No. of patientscensored (n, %) 3 (50.0) 5 (83.3) 5 (83.3) 5 (83.3) 15 (83.3) Cumulativeincidence (%) of relapse 33.3 0.0 16.7 16.7 11.1 at 1 year post-HCT (95%CI) [1] (2.9, 71.1) (NE) (0.5, 54.9) (0.5, 54.9) (1.7, 30.4) Treatmentcomparison: CD24Fc versus placebo [2] Hazard ratio (90% CI) 0.3 (0.1,1.4) Note: One year = Day 365 (+14 days) post-transplant (ie, Study Day380). Percentage was calculated using the number of patients in thecolumn heading as the denominator. [1] The cumulative incidence (%) ofrelapse 1 year post-HCT and the 95% CI were estimated using thecumulative incidence function with death without relapse as a competingrisk. For Day 365, if the maximum observed time was < Study Day 380, thecumulative incidence at the maximum observed time is presented for atreatment group. [2] Hazard ratio and 90% CI were based on a Fine andGray model with treatment as a covariate and death without relapse as acompeting risk. CI = confidence interval; HCT = hematopoietic stem celltransplantation; NE = not estimable; No. = number.

Graft-Versus-Host Disease-Free Survival and Relapse-Free Survival (GRFS)1 Year Following Hematopoietic Stem Cell Transplantation

This GRFS through 1 year post-HCT is a composite endpoint in whichevents included Grade III to IV acute GVHD, chronic GVHD requiringsystemic immunosuppressive therapy, relapse, or death from any cause.Table 16 summarizes Grade III to IV acute GRFS 1 year post-HCT for themITT Population.

The Kaplan-Meier estimate of the median GRFS (with 95% CI) was 229.0days (141.0, not estimable) for the overall CD24Fc treatment group:247.0 days (129.0, not estimable) for the 240 mg CD24Fc single dosecohort, 287.0 (24.0, not estimable) for the 480 mg CD24Fc single dosecohort, and 193.5 (100.0, not estimable) for the 960 mg CD24Fc multipledose cohort. The Kaplan-Meier estimate of the median GRFS (with 95% CI)was 120.0 days (46.0, not estimable) for the placebo group. Overall, theGRFS rate at 1 year post-HCT (with 95% CI) was 32.4% (12.7%, 54.0%) forthe CD24Fc treatment group and 33.3% (4.6%, 67.6%) for the placebogroup. The hazard ratio (with 90% CI) for CD24Fc versus placebo was 0.7(0.3, 1.7). Patients who were alive and had no documented occurrence ofGrade III to IV acute GVHD, chronic GVHD requiring systemicimmunosuppressive therapy, or relapse at the data cutoff date werecensored at the last assessment date.

TABLE 16 Graft-Versus-Host Disease-Free Survival and Relapse-FreeSurvival 1 Year Following Hematopoietic Stem Cell Transplantation - mITTPopulation CD24Fc CD24Fc CD24Fc 240 mg 480 mg 960 mg Single SingleMultiple CD24Fc Placebo Dose Dose Doses Total Statistic (N = 6) (N = 6)(N = 6) (N = 6) (N = 18) No. of patients with events (n, %) 4 (66.7) 4(66.7) 4 (66.7) 4 (66.7) 12 (66.7) Earliest contributing event AcuteGVHD (Grade III-IV) 1 (16.7) 0 (0.0) 1 (16.7) 0 (0.0) 1 (5.6) ChronicGVHD requiring 0 (0.0) 3 (50.0) 2 (33.3) 3 (50.0) 8 (44.4) systemicimmunosuppressive therapy Relapse 2 (33.3) 0 (0.0) 1 (16.7) 1 (16.7) 2(11-1) Death 1 (16.7) 1 (16.7) 0 (0.0) 0 (0.0) 1 (5.6) No. of patientscensored (n, %) 2 (33.3) 2 (33.3) 2 (33.3) 2 (33.3) 6 (33.3) GVHD-freesurvival and relapse-free survival (days) Kaplan-Meier estimate [1]Median (95% CI) 120.0 (46.0, NE) 247.0 (129.0, NE) 287.0 (24.0, NE)193.5 (100.0, NE) 229.0 (141.0, NE) Mean (SD) [2] 178.7 (151.48) 260.5(99.54) 250.3 (149.68) 227.2 (123.63) 246.0 (119.19) Median [2] 120.0247.0 287.0 193.5 229.0 Min, max [2] 46, 371+ 129, 378+ 24, 380+ 100,376+ 24, 380+ Treatment comparison: CD24Fc versus placebo [3] Hazardratio (90% CI) 0.7 (0.3, 1.7) Rate (%) of being alive without 33.3 (4.6,67.6) 33.3 (4.6, 67.6) 33.3 (4.6, 67.6) 33.3 (4.6, 67.6) 32.4 (12.7,54.0) GVHD or relapse at 1 year post-HCT (95% CI) [4] Note: One year =Day 365 (+14 days) post-transplant (ie, Study Day 380). Percentage wascalculated using the number of patients in the column heading as thedenominator. [1] The 95% CI for median was computed using the Brookmeyerand Crowley method with log-log transformation. [2] Censoring wasignored in the calculation for mean (SD) and median. A “+” after the minor max indicates a censored observation. [3] Hazard ratio and 90% CIwere based on a Cox proportional hazards model with treatment as acovariate. [4] Kaplan-Meier estimate. CI = confidence interval; GVHD =graft-versus-host disease; HCT = hematopoietic stem celltransplantation; log = logarithm; max = maximum; min = minimum; NE = notestimable; No. = number; SD = standard deviation.

Incidence of Non-Relapse Mortality 1 Year Following Hematopoietic StemCell Transplantation

Table 17 summarizes the cumulative incidence of NRM 1 year post-HCT forthe mITT Population. Overall, the cumulative incidence rate of NRM at 1year (with 95% CI) was 5.6% (0.3%, 23.1%) for the CD24Fc treatment groupand 16.7% (0.5%, 54.9%) for the placebo group. The hazard ratio (with90% CI) for CD24Fc versus placebo was 0.3 (0.0, 2.8). Patients who werealive at the end of the follow-up period (Day 365 [1 year]) withoutrelapse were censored at the last date they were known to be alive. Atleast 50.0% of patients in each treatment group were censored. Thecumulative incidence rate of NRM at Day 180 (with 95% CI) was 0.0% forthe CD24Fc treatment group and 16.7% (0.5%, 54.9%) for the placebogroup.

TABLE 17 Cumulative Incidence of Non-Relapse Mortality 1 Year FollowingHematopoietic Stem Cell Transplantation - mITT Population CD24Fc CD24FcCD24Fc 240 mg 480 mg 960 mg Single Single Multiple CD24Fc Placebo DoseDose Doses Total Statistic (N = 6) (N = 6) (N = 6) (N = 6) (N = 18) No.of patients who died without 1 (16.7) 1 (16.7) 0 (0.0) 0 (0.0) 1 (5.6)relapse by 1 year post-HCT (n, %) No. of patients with relapse by 2(33.3) 0 (0.0) 1 (16.7) 1 (16.7) 2 (11-1) 1 year post-HCT (n, %) No. ofpatients censored (n, %) 3 (50.0) 5 (83.3) 5 (83.3) 5 (83.3) 15 (83.3)Cumulative incidence (%) of 16.7 (0.5, 54.9) 16.7 (0.5, 54.9) 0.0 (NE)0.0 (NE) 5.6 (0.3, 23.1) NRM at 1 year post-HCT (95% CI) [1] Treatmentcomparison: CD24Fc versus placebo [2] Hazard ratio (90% CI) 0.3 (0.0,2.8) Note: One year = Day 365 (+14 days) post-transplant (ie, Study Day380). Percentage was calculated using the number of patients in thecolumn heading as the denominator. [1] The cumulative incidence (%) ofNRM at 1 year post-HCT and the 95% CI were estimated using thecumulative incidence function with relapse as a competing risk. For Day365, if the maximum observed time was < Study Day 380, the cumulativeincidence at the maximum observed time is presented for a treatmentgroup. [2] Hazard ratio and 90% CI were based on a Fine and Gray modelwith treatment as a covariate and relapse as a competing risk. CI =confidence interval; HCT = hematopoietic stem cell transplantation; NE =not estimable; No. = number; NRM = non-relapse mortality.

Incidence of Chronic Graft-Versus-Host Disease 1 Year FollowingHematopoietic Stem Cell

Table 18 summarizes the cumulative incidence of chronic GVHD 1 yearpost-HCT for the mITT Population. Overall, the cumulative incidence rateof chronic GVHD at 1 year post-HCT (with 95% CI) was 63.3% (34.1%,82.4%) for the CD24Fc treatment group and 33.3% (2.5%, 72.0%) for theplacebo group. The hazard ratio (with 90% CI) for CD24Fc versus placebowas 2.1 (0.6, 7.4). There were 3 moderate chronic GVHD in the 240 mgCD24Fc single dose cohort, 3 mild and 1 moderate chronic GVHD in the 480mg CD24Fc single dose cohort, and 2 mild and 3 moderate chronic GVHD inthe 960 mg CD24Fc multiple doses cohort. Two patients had mild chronicGVHD in the placebo group. Overall, there were no instances of severechronic GVHD. Patients who were alive and did not experience chronicGVHD at the end of the follow-up period (Day 365 [1 year]) were censoredat the last date of evaluation.

TABLE 18 Cumulative Incidence of Chronic Graft-Versus-Host Disease 1Year Following Hematopoietic Stem Cell Transplantation - mITT PopulationCD24Fc CD24Fc CD24Fc 240 mg 480 mg 960 mg Single Single Multiple CD24FcPlacebo Dose Dose Doses Total Statistic (N = 6) (N = 6) (N = 6) (N = 6)(N = 18) No. of patients with chronic GVHD 2 (33.3) 3 (50.0) 3 (50.0) 5(83.3) 11 (61.1) by 1 year post-HCT (n, %) No. of patients who diedwithout 3 (50.0) 1 (16.7) 1 (16.7) 1 (16.7) 3 (16.7) chronic GVHD by 1year post-HCT (n, %) No. of patients censored (n, %) 1 (16.7) 2 (33.3) 2(33.3) 0 (0.0) 4 (22.2) Cumulative incidence (%) of 33.3 50.0 50.0 83.363.3 chronic GVHD at 1 year post-HCT [1] 95% CI (2.5, 72.0) (7.0, 83.5)(6.9, 83.6) (0.5, 99.4) (34.1, 82.4) Treatment comparison: CD24Fc versusplacebo [2] Hazard ratio (90% CI) 2.1 (0.6, 7.41 Note: One year = Day365 (+14 days) post-transplant (ie, Study Day 380). Percentage wascalculated using the number of patients in the column heading as thedenominator. [1] The cumulative incidence (%) of chronic GVHD at 1 yearpost-HCT and the 95% CI were estimated using the cumulative incidencefunction with death without chronic GVHD as a competing risk. If themaximum observed time was < Study Day 380, the cumulative incidence atthe maximum observed time is presented for a treatment group. [2] Hazardratio and 90% CI were based on a Fine and Gray model with treatment as acovariate and death without chronic GVHD as a competing risk. CI =confidence interval; GVHD = graft-versus-host disease; HCT =hematopoietic stem cell transplantation; No. = number.

Rate of Infection at Day 100

As with the effect on GVL, therapeutic strategies designed to preventGVHD through global immune suppression may result in an increase ininfection rates, including bacterial infections and CMV reactivation.

Table 19 summarizes the incidence of infections through Day 100 for themITT Population. In total, 13 (72.2%) patients who received CD24Fc (5[83.3%] patients in the 240 mg CD24Fc single dose cohort, 2 [33.3%]patients in the 480 mg CD24Fc single dose cohort, and 6 [100.0%]patients in the 960 mg CD24Fc multiple dose cohort) and 2 (33.3%)patients who received placebo had an infection through Day 100.

Most infections were considered to be controlled and resolved. Patient103-001 in the placebo group died from pneumonia. Patient 102-002 in theplacebo group had conjunctivitis that was reported asrecovering/resolving. Patient 101-010 in the 480 mg CD24Fc single dosecohort and Patient 101-011 in the 480 mg CD24Fc single dose cohort bothhad rash pustular that was reported as not recovered/not resolved.Patient 102-006 in the 960 mg CD24Fc multiple dose cohort had upperrespiratory tract infection and Clostridium difficile colitis that werereported as intervention continued.

The majority of the infections were bacterial (9 [50.0%] patients whoreceived CD24Fc and 2 [33.3%] patients who received placebo) or viral (7[38.9%] patients who received CD24Fc and 1 [16.7%] patient who receivedplacebo). The majority of infections occurred in the blood (8 [44.4%]patients who received CD24Fc and 1 [16.7%] patient who receivedplacebo), urine (4 [22.2%] patients who received CD24Fc and no patientswho received placebo), or feces (2 [11.1%] patients who received CD24Fcand 2 [33.3%] patients who received placebo). The majority of thebacteria recovered from blood culture were common skin inhabitants andlow virulence pathogens (ie, coagulase negative staphylococci).

TABLE 19 Summary of Incidence of Infections Through Day 100 - mITTPopulation CD24Fc CD24Fc CD24Fc 240 mg 480 mg 960 mg Single SingleMultiple CD24Fc Placebo Dose Dose Doses Total (N = 6) (N = 6) (N = 6) (N= 6) (N = 18) Statistic n (%) n (%) n (%) n (%) n (%) No. of patientswith any infections 2 (33.3) 5 (83.3) 2 (33.3) 6 (100.0) 13 (72.2)through Day 100 Type of infection Bacterial 2 (33.3) 2 (33.3) 2 (33.3) 5(83.3) 9 (50.0) Fungal 0 (0.0) 0 (0.0) 2 (33.3) 0 (0.0) 2 (11.1) Viral 1(16.7) 3 (50.0) 1 (16.7) 3 (50.0) 7 (38.9) Site of infection Blood 1(16.7) 2 (33.3) 2 (33.3) 4 (66.7) 8 (44.4) Disseminated (2 or moresites) 0 (0.0) 0 (0.0) 0 (0.0) 1 (16.7) 1 (5.6) Feces 2 (33.3) 1 (16.7)0 (0.0) 1 (16.7) 2 (11.1) Gastrointestinal system 0 (0.0) 0 (0.0) 0(0.0) 1 (16.7) 1 (5.6) Genitalia 0 (0.0) 0 (0.0) 1 (16.7) 0 (0.0) 1(5.6) Lower respiratory system 1 (16.7) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0)Skin 0 (0.0) 0 (0.0) 1 (16.7) 1 (16.7) 2 (11.1) Upper respiratory system0 (0.0) 1 (16.7) 0 (0.0) 2 (33.3) 3 (16.7) Urine 0 (0.0) 1 (16.7) 1(16.7) 2 (33.3) 4 (22.2) Percentage was calculated using the number ofpatients in the column heading as the denominator. No. = number.

As shown in Table 20, there were 9 patients in the CD24Fc group that hadhigh risk of CMV reactivation (Donor/Recipient CMV status before HCT:D+/R+, 5; D−/R+, 3; unknown D/R+, 1). One patient in the CD24Fc groupwith D+/R− had intermediate risk for CMV reactivation. Eight patients inthe CD24Fc group had status of D−/R−, which was considered to be lowrisk. Two D−/R+ patients had CMV reactivation at Day 42 and Day 48,representing 22.2% cumulative incidence of CMV reactivation at Day 100in the high risk group. Both patients had systemic steroid treatmentprior to the detection of CMV reactivation. In comparison, 2 patients inthe placebo group were high risk of CMV reactivation (D+/R+, 1; D−/R+,1). One patient in the placebo group had CMV reactivation at Day 47before systemic steroid treatment for acute GVHD (50.0% in high riskgroup).

CMV infection rates in HCT patients stratified by donor and recipientCMV status before transplant. D=donor, R=recipient, +is positive, − isnegative, U is unknown.

Cytomegalovirus status CD24Fc Group Placebo Group D+, R+ 5 1 D+, R− 1 0D−, R+ 3 1 D−, R− 8 4 DU, R+ 1 0

Overall, CD24Fc was well tolerated in the phase IIa study. There were noinfusion-related toxicities. There was one possible drug relatedTEAE≥grade III in patients exposed to CD24Fc in the 480 mg group ofhyperglycemia, which was managed with insulin. One dose-limitingtoxicity (DLT) was observed in the placebo group, and no DLTs wereobserved in the CD24Fc groups. There were no adverse events leading todeath in patients administered CD24Fc within the 180 days (at least 150days after the last dosing of CD24Fc). There was one adverse event ofpneumonia that led to the death of a subject at Day 48 in the placebogroup. One patient in CD24Fc group died 7 months after HCT, though thedeath was determined to be unlikely related to study drug. Anti-drugantibodies (ADA) were not detected in any of the 24 patients at anypoint out to day 100 after HCT.

The most common TEAEs≥grade III (>10%) included a decrease in plateletcounts (83.3% placebo and 94.4% CD24Fc), decrease in WBC counts (66.7%placebo and 88.9% CD24Fc), decrease in neutrophil counts (50% placeboand 83.3% CD24Fc), decrease in lymphocyte counts (50% placebo and 77.8%CD24Fc), anemia (50% placebo and 66.7% CD24Fc), stomatitis (83.3%placebo and 50% CD24Fc), and nausea (0% placebo and 11.1% CD24Fc). TheseSAEs are consistent with the known safety profile of myeloablativeconditioning regimens used in HCT.

Myeloablative conditioning for HCT is often associated with severeregimen related toxicity including organ failure. Organ failure is themost frequent cause of early onset transplantation related mortality(TRM) or non-relapse mortality (NRM). In the CD24Fc group of 18patients, none of the patients died within the first 100 days post HCT,while 1 out of 6 in the placebo group died on Day 48 due to respiratoryfailure.

Pharmacokinetic Results

FIGS. 22-23 show the PK data from the three escalation cohorts from thePhase 2a trial. The half-life from the 240 and 480 mg single dosecohorts (FIG. 22) was around about 14 days, which is consistent with thedata seen in healthy subjects. At 480 mg there was higher cmx but noreal increase in exposure after 14 days, the time of peek GVHD incidenceand engraftment. In the final multi-dose cohort there was increasedexposure through day 60 as expected (FIG. 23), the period during whichpatients are most susceptible to develop GVHD.

Table 21 summarizes the plasma PK parameters of CD24Fc for the PKPopulation in the single dose cohorts. The geometric mean C_(max,−1 d)values were 52,145.41 and 84,155.08 ng/mL, the geometric meanAUC_(0-last,−1 d) values were 10,156,549.9 and 15,522,686.2 ng h/mL, thegeometric mean AUC_(0-42 d) values were 9,275,562.3 and 13,903,718.4 ngh/mL, and the geometric mean AUC_(0-inf) values were 10,383,503.9 and15,716,616.4 ng h/mL for the 240 and 480 mg CD24Fc single dose cohorts,respectively. Median t_(max,−1 d) was 2.10 h for both the 240 and 480 mgCD24Fc single dose cohorts. The mean values of t½ were 414.739 and406.648 h and the mean values of λz were 0.0018 and 0.0017 h−1 for the240 and 480 mg CD24Fc single dose cohorts, respectively. The mean Vzvalues were 13.83 and 18.18 L, and the mean CL values were 0.024 and0.031 L/h for the 240 and 480 mg CD24Fc single dose cohorts,respectively.

TABLE 20 Summary of Plasma Pharmacokinetic Parameters of CD24Fc - PKPopulation - Single Dose Cohorts CD24Fc CD24Fc 240 mg 480 mg Single DoseSingle Dose PK Parameter (Unit) N Statistic N Statistic C_(max, −1 d)(ng/mL) [1] 6 52,145.41 (22.3) 6 84,155.08 (24.6) t_(max, −1 d) (h) [2]6 2.10 (2.1, 2.4) 6 2.10 (2.0, 2.2) AUC_(0-42 d) (ng · h/mL) [1] 69,275, 562.3 (23.2) 6 13,903, 718.4 (19.7) AUC_(0-last, −1 d) (ng ·h/mL) [1] 6 10,156, 549.9 (26.2) 6 15,522,686.2 (21.3) AUC_(0-inf) (ng ·h/mL) [1] 6 10,383, 503.9 (25.2) 6 15,716, 616.4 (21.5) AUC_(extrap) (%)[3] 6 2.17 (1.669) 6 1.23 (0.519) λ₂ (h⁻¹) [3] 6 0.0018 (0.0005) 60.0017 (0.0002) t_(1/2) (h) [3] 6 414.739 (110.4483) 6 406.648 (62.3044)V_(z) (L) [3] 6 13.83 (3.586) 6 18.18 (4.529) CL (L/h) [3] 6 0.024(0.0059) 6 0.031 (0.0071) Geometric CV % = 100*(exp(SD²) − 1)^(0.5),where SD is the standard deviation of the logarithm-transformed data.[1] Geometric mean (geometric CV %) [2] Median (minimum, maximum) [3]Mean (SD) λ₂ = apparent terminal elimination rate constant; AUC_(0-42 d)= the area under the plasma concentration versus time curve from time 0to Day 42; AUC_(0-inf) = the area under the concentration versus timecurve from time 0 extrapolated to infinity; AUC_(0-last, −1 d) = thearea under the plasma concentration versus time curve from time 0 to thelast measurable plasma drug concentration for Day −1 dosing;AUC_(extrap) = percentage of AUC_(0-inf) that was due to extrapolationfrom the last measurable plasma drug concentration to infinity; CL =total body clearance after intravenous administration; C_(max, −1 d) =maximum observed plasma concentration for Day −1 dosing; CV % =coefficient of variation; PK = pharmacokinetic; SD = standard deviation;t_(1/2) = apparent terminal elimination half-life; t_(max, −1 d) = thetime of maximum observed plasma concentration for Day −1 dosing; V_(z) =volume of distribution based on the terminal elimination phase.

Table 22 summarizes the plasma PK parameters of CD24 Fc for the PKpopulation in the multiple dose cohort on Day −1, Day 28, and Day −1 toDay 100. The geometric mean C_(max,−1 d) and C_(max,28 d) values were96,942.71 ng/mL and 62,563.05 ng/mL, respectively, for the 960 mg CD24Fcmultiple dose cohort. The geometric mean AUC_(0-last,−1 d),AUC_(0-14 d), AUC_(0-100 d), and AUC_(0-last), overall values were12,317,971.2 ng h/mL, 9,688,933.9 ng h/mL, 37,736,555.1 ng h/mL, and37,363,953.5 ng h/mL, respectively, for the 960 mg CD24Fc multiple dosecohorts. The median t_(max,−1 d) and t_(max,28 d) were 2.13 h and 2.52h, respectively, for the 960 mg CD24Fc multiple dose cohort.

TABLE 21 Summary of Plasma Pharmacokinetic Parameters of CD24Fc - PKPopulation - Multiple Dose Cohort - Day −1, Day 28, and Day −1 to Day100 CD24Fc CD24Fc CD24Fc 960 mg 960 mg 960 mg Multiple Doses MultipleDoses Multiple Doses Day −1 Day 28 Day −1 to Day 100 PK Parameter (Unit)N Statistic N Statistic N Statistic C_(max, −1 d) (ng/mL) [1] 696,942.71 (41.5) — — — — t_(max, −1 d) (h) [2] 6 2.13 (2.0, 3.2) — — — —AUC_(0-last, −1 d) (ng · h/mL) [1] 6 12,317,971.2 (24.9) — — — —C_(max, 28 d) (ng/mL) [1] — — 6 62,563.05 (34.1) — — t_(max, 28 d) (h)[2] — — 6 2.52 (2.0, 4.0) — — C_(min) (ng/mL) [1] — — 6 13,233.79 (33.6)— — T_(min) (h) [2] — — 6 0.00 (0.0, 308.2) — — AUC_(0-14 d) (ng · h/mL)[1] — — 6 9,688,933.9 (30.9) — — C_(avg) (ng/mL) [1] — — 6 28,836.11(30.9) — — Cl_(ss) (L/h) [3] — — 6 0.026 (0.0078) — —AUC_(0-last, overall) (ng · h/mL) [1] — — — — 6 37,363,953.5 (27.6)AUC_(0-100 d) (ng · h/mL) [1] — — — — 6 37,736,555.1 (29.3) Geometric CV% = 100*(exp(SD²) − 1)^(0.5), where SD is the standard deviation of thelogarithm-transformed data. [1] Geometric mean (geometric CV %) [2]Median (minimum, maximum) [3] Mean(SD) AUC_(0-14 d) = the area under theplasma concentration versus time curve from time 0 to Tau; AUC_(0-100 d)= the area under the concentration versus time curve from time 0 on Day−1 to Day 100; AUC_(0-last, −1 d) = the area under the plasmaconcentration versus time curve from time 0 to the last measurableplasma drug concentration for Day −1 dosing; AUC_(0-last, overall) = thearea under the plasma concentration versus time curve from time 0 on Day−1 to the last measurable plasma drug concentration after the last doseon Day 28; C_(avg) = calculated as AUC_(0-14 d) divided by Tau; Cl_(ss)= calculated as Dose/AUC_(0-14 d); C_(max, 1 d) = maximum observedplasma concentration for Day −1 dosing; C_(max, 28 d) = maximum observedplasma concentration between dose time and dose time + Tau for Day 28dosing; C_(min) = minimum concentration between dose time and dosetime + Tau (at the time of minimum concentration sampled during a dosinginterval); CV % = coefficient of variation; PK = pharmacokinetic; SD =standard deviation; t_(max, −1 d) = the time of maximum observed plasmaconcentration for Day −1 dosing; t_(max, 28 d) = the time of maximumobserved plasma concentration during a dosing interval for Day 28dosing; T_(min) = time of minimum concentration sampled during a dosinginterval.

The clinical evidence from the phase IIa study strongly suggests thatCD24Fc, administered in combination with methotrexate and tacrolimus,greatly improves outcomes in leukemia patients undergoing myeloablativeallo-HCT by reducing both the likelihood of severe aGVHD (grades III-IV)and the likelihood of leukemia relapse. As described above, thecumulative incidence of grade III-IV aGVHD is 5.6% in CD24Fc exposedpatients as compared to 16.7% in the placebo cohort (saline plusmethotrexate and tacrolimus) and 24% in the contemporary control cohort(methotrexate and tacrolimus alone). These data suggest thatadministration of CD24Fc in combination with methotrexate and tacrolimusas prophylaxis reduces the risk of grade III-IV aGVHD in HCT patients,the most serious grades of aGVHD which are associated with increasedrisk of non-relapse mortality. A trend of reduction is observed in theincidence of relapse in patients who received CD24Fc (11.1%) as comparedto patients who did not, both as compared to the placebo arm (33.3%) andthe contemporary control (23%), demonstrating that CD24Fc does notaffect the GVT effects of the graft and may even reduce the risk ofleukemia relapse. The benefit of including CD24Fc in standard GVHDprophylaxis regimens is further supported by the better NRM in CD24Fcexposed patients (5.6%) as compared to placebo (16.7%), better 1.5-yearoverall survival (89% versus 50%, CD24Fc versus placebo control), astatistically significant improvement in grade III-IV aGVHD RFS (83%versus 33%, CD24Fc versus placebo control, respectively), adose-dependent reduction in severe mucositis, and a good safety profilewith only one drug-related TEAE (grade III) observed in the study.

A prophylaxis agent that reduces the risk of both aGVHD and leukemiarelapse would be novel and extremely beneficial to leukemia patientsundergoing allo-HCT following myeloablative conditioning. As describedabove, the early clinical data in this application strongly suggeststhat administration of CD24Fc in combination with methotrexate andtacrolimus provides a substantial improvement over existing prophylaxisregimens on the clinically significant endpoints of grade III-IV aGVHDprevention and leukemia relapse, and thus should be eligible forBreakthrough Designation. The effects of CD24Fc observed in the phaseIIa portion of the clinical study will be further investigated in thephase IIb portion, which has been designed to confirm the efficacy ofprophylactic CD24Fc administration in reducing Grade III-IV aGVHD andleukemia relapse in leukemia patients undergoing allo-HCT followingmyeloablative conditioning.

EXAMPLE 6 CD24 can be Used to Reduce Mucositis in Subjects UndergoingHCT

Myeloablative conditioning for HCT is often associated with severeregimen related toxicity including grade 3-4 mucositis. Severe oralmucositis has been reported by HCT patients as the most distressingsymptom they experienced. As a measure of the effect of CD24Fc treatmenton mucositis in subjects in the Phase IIa GVHD prophylaxis trialdescribed in Example 5, we generated a combined mucositis scoring systemto study the outcome. This data is shown in FIG. 24A and comprises themultiple of the number of days patients spent with serious (Grade 3 or4) mucositis. The score is provided in the bars, along with the numberof pts that had the mucositis in parentheses. Oral mucositis was scoredaccording to CTCAE 4.01. As shown in FIG. 24B, dose-dependent reductionsin the mucositis grade-day scores were observed across all CD24Fccohorts as compared to placebo (R=−0.9983; P=0.0009) and a statisticallysignificant reduction was observed in the multi-dose cohort (placebo vs.960 mg multi-dose: P=0.035, Student's t-test).

1. (canceled)
 2. (canceled)
 3. A method of treating or preventingmucositis in a subject in need thereof, comprising administering a CD24protein to the subject.
 4. The method of claim 3, wherein the subjecthas a cancer.
 5. The method of claim 4, wherein the cancer is AcuteMyeloid Leukemia (AML), Acute Lymphoblastic Leukemia (ALL), ChronicMyelogenous Leukemia (CML), Myelodysplastic syndrome (MDS), or ChronicMyelomonocytic Leukemia (CMML).
 6. The method of claim 3 wherein theCD24 protein is administered at a dose of 240 mg or 480 mg.
 7. Themethod of claim 3, wherein the subject will undergo or has undergone ahematopoietic stem cell transplantation (HCT).
 8. The method of claim 7,wherein the CD24 protein is administered before or after the HCT.
 9. Themethod of claim 8, wherein the CD24 protein is administered one daybefore the HCT.
 10. The method of claim 8, wherein the CD24 protein isadministered more than once.
 11. The method of claim 10, wherein theCD24 protein is administered in three biweekly doses, comprising a doseon the day before the HCT, a dose on day 14 after the HCT, and a dose onday 28 after the HCT
 12. The method of claim 11, wherein the doses ofthe CD24 protein are 480 mg, 240 mg, and 240 mg, respectively.
 13. Themethod of claim 3, wherein the CD24 protein comprises a mature humanCD24 polypeptide fused at its N-terminus or C-terminus to a Fc region ofa mammalian immunoglobulin (Ig) protein.
 14. The method of claim 13,wherein the mature human CD24 polypeptide comprises the sequence setforth in SEQ ID NO: 1 or
 2. 15. The method of claim 14, wherein the Igprotein is human, and wherein the Fc region comprises a hinge region andCH2 and CH3 domains of IgG1, IgG2, IgG3, IgG4, or IgA.
 16. The method ofclaim 14, wherein the Ig protein is human, and wherein the Fc regioncomprises a hinge region and CH2, CH3 and CH4 domains of IgM.
 17. Themethod of claim 15, wherein the CD24 protein comprises the sequence setforth in SEQ ID NO: 6, 11, or
 12. 18. The method of claim 17, whereinthe amino acid sequence of the CD24 protein consists of the sequence setforth in SEQ ID NO: 6, 11, or
 12. 19. The method of claim 3, wherein theCD24 protein is soluble and/or glycosylated.
 20. (canceled)
 21. Themethod of claim 3, wherein the CD24 protein is prepared using aeukaryotic expression system.
 22. The method of claim 21, wherein theeukaryotic expression system comprises expression from a vector inmammalian cells.
 23. The method of claim 22, wherein the mammalian cellsare Chinese Hamster Ovary cells. 24.-46. (canceled)